CD27L antigen binding proteins

ABSTRACT

The present invention relates to CD27L antigen binding proteins, such an antibodies, polynucleotides encoding said CD27l antigen binding proteins, antibody drug conjugate compositions, and methods for diagnosing and treating diseases associated with CD27L expression.

RELATED APPLICATIONS

This application is a divisional application of and claims benefit ofU.S. Non-Provisional application Ser. No. 13/632,836 filed on Sep. 20,2012, which application in turn claims the benefit of U.S. ProvisionalApplication No. 61/538,024 filed on Sep. 22, 2011, both specificationsof which are hereby incorporated herein by reference in their entirety.

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-1437-US-NP (US Non-Prov)_ST25.txt, created Sep. 17, 2012, which is94.3 KB in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The field of this invention relates to compositions of antigen bindingproteins including antibodies capable of binding CD27L, as well asrelated methods.

BACKGROUND

CD27L (CD70, TNFSF7) is a type II integral membrane protein whoseexpression on normal tissues is highly restricted to a subset ofactivated T and B cells, dendritic cells and to a small subset of cellsin the thymic epithelium. The biological functions of CD27L, whichinclude augmentation or regulation of the immune response, are mediatedvia binding to its receptor, CD27, which is expressed predominately onlymphoid cells. CD27L/CD27 interactions regulate B-cell proliferationand differentiation and T-cell costimulation/activation. Disruption ofthe CD27L/CD27 interaction in mice deficient for CD27 does not result inany phenotype in the absence of an immune challenge. (Grewal, ExpertOpin. Ther. Targets. 12, 341-351 (2008)).

In addition to its very restricted expression on normal tissues, CD27Lis expressed at relatively high levels in some B cell non-Hodgkin'slymphoma (B-NHL) tumor sub-types, in pre-B cell acute lymphocyticleukemia (ALL) and in B cell type-chronic lymphocytic leukemia (B-CLL).Aberrant expression of CD27L is also observed in renal cell carcinoma(RCC) but not in normal kidney or other normal tissues. Thus, CD27Lcomes close to exhibiting properties consistent with those of a “tumorspecific antigen” (Grewal, Expert Opin. Ther. Targets. 12, 341-351(2008)).

Each year, of the approximately 49,000 patients that will develop RCC, alittle over 40,000 of those will be diagnosed with ccRCC in the US(American Cancer Society: Cancer Facts and Figures final (2008). Whilesome newer therapeutics have been approved for RCC over the last 4years, the 5 year survival rate for patients with metastatic RCC remainsdismal at 10-20% (National Cancer Institute. SEER cancer statistics factsheet: cancer of the kidney and renal pelvis—accessed 2008) andsignificant unmet medical need remains. The projected yearly number ofnewly diagnosed ccRCC patients (U.S.) that are expected to express CD27Lis approximately 36,000. There are an estimated 64,000 ccRCC patientscurrently with active disease.

Of the B-cell malignancies reported to aberrantly express CD27L, theB-NHL subsets of diffuse large cell B-cell lymphoma (DLBCL) andfollicular lymphoma (FL) show the highest incidence of expressionranging from 33% for FL to 71% for DLBCL as assessed by IHC on frozensections using a validated antibody (Lens et al., Brit. J. Hematol. 106,491-503 (1999). 50-89% of B-CLL tumors also express CD27L as assessed byIHC on frozen tumor sections or by flow cytometry on circulating tumorcells (Ranheim et al., Blood 85, 3556-3565 (1965); Trentin et al.,Cancer Res. 57, 4940-4947 (1997)).

Of the 127,000 patients in the US currently with active B-NHL,approximately 50% of these patients present with the DLBCL (intermediategrade) sub-type (Morton et al., Blood 107, 265-276 (2002)). DespiteRituxan plus cyclophosphamide, adriamycin, vincristine, prednisone(CHOP) standard of care therapy for DLBCL patients, almost 50% relapse.Therefore an unmet medical need remains in this disease as well.

SUMMARY

The invention provides anti-CD27L antigen binding proteins, e.g.,antibodies and functional fragments thereof. The anti-CD27L antigenbinding proteins are particularly useful in methods of treating diseasesand disorders associated with aberrant cell proliferation, e.g., cancer,and/or with inflammation.

In a first aspect, the CD27L antigen binding protein comprises a) alight chain variable domain having at least 90% identity, at least 95%identity, or is identical to the amino acid sequence set forth in SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, or SEQ ID NO:70; b) a heavy chain variable domainhaving at least 90% identity, at least 95% identity, or is identical tothe amino acid sequence set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24; or c) the light chain variable domain of a) and the heavy chainvariable domain of b).

Preferred antigen binding proteins of the first aspect include thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:63 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:17; those comprising a light chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:64 and a heavy chain variable domain having at least 90%,at least 95%, or is identical to the amino acid sequence set forth inSEQ ID NO:18; those comprising a light chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:65 and a heavy chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:19; those comprising a light chain variable domain havingat least 90%, at least 95%, or is identical to the amino acid sequenceset forth in SEQ ID NO:66 and a heavy chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:20; those comprising a light chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:67 and a heavy chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:21; those comprising a light chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:68 and a heavy chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:22; those comprising alight chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:69 and aheavy chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:23; andthose comprising a light chain variable domain having at least 90%, atleast 95%, or is identical to the amino acid sequence set forth in SEQID NO:70 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:24.

In a second aspect, the CD27L antigen binding protein comprises a) alight chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70; b) aheavy chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; or c)the light chain variable domain of a) and the heavy chain variabledomain of b).

Preferred antigen binding proteins of the second aspect include thosecomprising a light chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:63 and a heavy chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:17; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:64 and a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:18; those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:65 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:19;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:66 and a heavy chainvariable domain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:20; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:67 and a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:21; those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:68 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:22;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:69 and a heavy chainvariable domain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:23; and those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:70 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:24.

In a third aspect, the CD27L antigen binding protein contains a lightchain variable domain comprising a) an LCDR1 having no more than threeamino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:71; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:79; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:87; b) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:72; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:80; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:88; c) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:73; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:81; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:89; d) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:74; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:82; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:90; e) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:75; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:83; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:91; f) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:76; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:84; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:92; g) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:77; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:85; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:93; or h) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:78; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:86; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:94; and a heavy chain variable domaincomprising i) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:25; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:33; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:41; j) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:26; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:34; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:42; k) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:27; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:35; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:43; l) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:28; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:36; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:44; m) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:29; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:37; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:45; n) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:30; an HCDR2 having Gno more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:38; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:46; o) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:31; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:39; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:47; or p) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:32; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:40; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:48.

Preferred CD27L antigen binding proteins of third aspect include thosecomprising the light chain variable domain of a) and the heavy chainvariable domain of i); those comprising the light chain variable domainof b) and the heavy chain variable domain of j); those comprising thelight chain variable domain of c) and the heavy chain variable domain ofk); those comprising the light chain variable domain of d) and the heavychain variable domain of l); those comprising the light chain variabledomain of e) and the heavy chain variable domain of m); those comprisingthe light chain variable domain of f) and the heavy chain variabledomain of n); those comprising the light chain variable domain of g) andthe heavy chain variable domain of o); and those comprising the lightchain variable domain of h) and the heavy chain variable domain of p).

In a fourth aspect of the invention, the CD27L antigen binding proteinof the first, second, or third aspect binds to human CD27L with anaffinity of less than or equal to 2×10⁻¹¹ M.

In a fifth aspect of the invention, the CD27L antigen binding protein ofthe first, second, third, or fourth aspect inhibits binding of CD27L toCD27.

In a sixth aspect of the invention, the CD27L antigen binding protein ofthe first, second, third, fourth, or fifth aspect is an antibody, suchas a human antibody. Preferred antibodies include those antibodies thatcomprise a light chain having the amino acid sequence set forth in SEQID:56 and a heavy chain having the amino acid sequence set forth in SEQID NO:10; those that comprise a light chain having the amino acidsequence set forth in SEQ ID:57 and a heavy chain having the amino acidsequence set forth in SEQ ID NO:11; those that comprise a light chainhaving the amino acid sequence set forth in SEQ ID:58 and a heavy chainhaving the amino acid sequence set forth in SEQ ID NO:12; those thatcomprise a light chain having the amino acid sequence set forth in SEQID:59 and a heavy chain having the amino acid sequence set forth in SEQID NO:13; those that comprise a light chain having the amino acidsequence set forth in SEQ ID:60 and a heavy chain having the amino acidsequence set forth in SEQ ID NO:14; those that comprise a light chainhaving the amino acid sequence set forth in SEQ ID:61 and a heavy chainhaving the amino acid sequence set forth in SEQ ID NO:15; and those thatcomprise a light chain having the amino acid sequence set forth in SEQID:62 and a heavy chain having the amino acid sequence set forth in SEQID NO:16.

In a seventh aspect, a CD27L antigen binding protein of the first,second, third, fourth, fifth, or sixth aspect is conjugated to a drug orchemotherapeutic agent. In preferred embodiments, the drug orchemotherapeutic agent is conjugated to the antigen binding protein,e.g. antibody, using a linker. Preferred linkers include non-cleavablelinkers such as MCC. A preferred chemotherapeutic agent is DM1. Thus, inparticularly preferred embodiments, the seventh aspect provides a CD27Lantigen binding protein of the first, second, third, fourth, fifth, orsixth aspect conjugated to DM1 by a MCC linker attached to one or morelysine residues.

The process of conjugating DM1 to a CD27L antigen binding protein, e.g.antibody, will produce a composition comprising a population ofDM1-conjugated antibodies having a range of DM1 molecules per antibody.It is possible to measure an average number for the composition. Inpreferred embodiments, the average number of DM1 molecules per CD27Lantigen binding protein, e.g. antibody, is between 1 and 10, between 3and 7, or between 4 and 6. In preferred embodiments, the composition ofCD27L antigen binding proteins of the first, second, third, fourth,fifth, sixth, or seventh aspect of this invention has an average numberof DM1 molecules per CD27L antigen binding protein of about 4.0, about4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7,about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about6.0. Such a composition may contain a therapeutically effective amountof the CD27L antigen binding protein and may be lyophilized.

In an eight aspect, the invention provides isolated nucleic acidsencoding one or more polypeptide components of a CD27L antigen bindingprotein, e.g., an antibody light chain or antibody heavy chain. Inpreferred embodiments the nucleic acid encodes a polypeptide comprising:

a) a light chain variable domain having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ IDNO:70;

b) a heavy chain variable domain having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24;

c) a light chain variable domain having no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70;d) a heavy chain variable domain having no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24;e) a light chain variable domain comprising:

-   -   i) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:71; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:79; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:87;    -   ii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:72; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:80; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:88;    -   iii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:73; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:81; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:89;    -   iv) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:74; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:82; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:90;    -   v) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:75; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:83; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:91;    -   vi) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:76; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:84; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:92;    -   vii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:77; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:85; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:93; or    -   viii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:78; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:86; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:94; or        f) a heavy chain variable domain comprising:    -   i) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:25; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:33; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:41;    -   ii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:26; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:34; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:42;    -   iii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:27; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:35; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:43;    -   iv) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:28; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:36; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:44;    -   v) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:29; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:37; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:45;    -   vi) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:30; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:38; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:46;    -   vii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:31; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:39; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:47; or    -   viii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:32; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:40; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:48.

In certain embodiments of the eighth aspect, the polypeptide encodes anantibody light chain and is at least 80%, at least 90%, at least 95%, oris 100% identical to the nucleotide sequence set forth in SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, orSEQ ID NO:55. In other embodiments of the eighth aspect, the polypeptideencodes an antibody heavy chain and is at least 80%, at least 90%, atleast 95%, or is 100% identical to the nucleotide sequence set forth inSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, or SEQ ID NO:9.

In a ninth aspect, the invention provides an expression vectorcomprising one or more isolated nucleic acids of the eighth aspect. Incertain embodiments, the expression vector encodes an antibody lightchain, an antibody heavy chain, or both an antibody light chain and aheavy chain.

In a tenth aspect, the invention provides a recombinant host cellcomprising one or more isolated nucleic acids of the eighth aspectoperably linked to a promoter, including recombinant host cellscomprising one or more expression vectors of the ninth aspect of theinvention. In preferred embodiments, the recombinant host cell secretesan antibody that binds CD27L. Preferred host cells are mammalian hostcells, including CHO cell lines.

In an eleventh aspect, the invention provides methods of making a CD27Lantibody drug conjugate of the seventh aspect by conjugating a linkerand drug, e.g., chemotherapeutic agent, to any of the CD27L antigenbinding proteins of the first, second, third, fourth, fifth, or sixthaspects. The linker and drug may be connect first and then conjugated tothe CD27L antigen binding protein or the linker may be first conjugatedto the CD27L antigen binding protein then connected to the drug. Inpreferred embodiments, the linker is MCC and the drug is DM1. Inparticularly preferred embodiments, the CD27L antigen binding protein isan antibody comprising a light chain variable domain amino acid sequenceas set forth in SEQ ID NO:63 and a heavy chain variable domain aminoacid sequence as set forth in SEQ ID NO:17 (e.g., Ab1), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:64 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:18 (e.g., Ab2), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:65 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:19 (e.g., Ab3), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:66 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:20 (e.g., Ab4), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:67 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:21 (e.g., Ab5), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:68 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:22 (e.g., Ab6), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:69 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:23 (e.g., Ab7), or an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:70 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:24 (e.g., Ab8) conjugated to DM1 byMCC by chemically reacting one or more lysine residues within theantibody with MCC or MCC-DM1.

In a twelfth aspect, the invention provides methods of treating cancercomprising administering to a patient a therapeutically effective amountof a composition comprising a therapeutically effective amount of aCD27L antigen binding protein of the first, second, third, fourth,fifth, or sixth aspect. In preferred embodiments, the CD27L antigenbinding protein is an antibody comprising a light chain variable domainamino acid sequence as set forth in SEQ ID NO:63 and a heavy chainvariable domain amino acid sequence as set forth in SEQ ID NO:17 (e.g.,Ab1), an antibody comprising a light chain variable domain amino acidsequence as set forth in SEQ ID NO:64 and a heavy chain variable domainamino acid sequence as set forth in SEQ ID NO:18 (e.g., Ab2), anantibody comprising a light chain variable domain amino acid sequence asset forth in SEQ ID NO:65 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:19 (e.g., Ab3), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:66 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:20 (e.g., Ab4), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:67 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:21 (e.g., Ab5), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:68 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:22 (e.g., Ab6), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:69 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:23 (e.g., Ab7), or an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:70 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:24 (e.g., Ab8). In particularlypreferred embodiments, the CD27L antigen binding protein is conjugatedto a chemotherapeutic agent (e.g., DM1) by a linker (MCC). In otherpreferred embodiments of the twelfth aspect, the antibody comprisesenhanced effector function.

In some embodiments, the CD27L antigen binding protein is administeredto a patient having renal cell carcinomas (RCC), clear cell RCC, headand neck cancer, glioblastoma, breast cancer, brain tumor,nasopharangeal carcinoma, non-Hodgkin's lymphoma (NHL), acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL),Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), multiplemyeloma, cutaneous T-cell lymphomas, nodular small cleaved-celllymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert'slymphomas, immunoblastic lymphoma, T-cell leukemia/lymphomas (ATLL),adult T-cell leukemia (T-ALL), entroblastic/centrocytic (cb/cc)follicular lymphoma cancer, diffuse large cell lymphoma of B lineage,angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma, HIVassociated body cavity based lymphoma, embryonal carcinoma,undifferentiated carcinoma of the rhino-pharynx, Castleman's disease,Kaposi's Sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia orother B-cell lymphoma.

In certain embodiments, a sample from a patient is tested for CD27Lexpression prior to administering the CD27L antigen binding protein.CD27L expression may be determined by testing for the presence ofCD27L-encoding RNA or for the presence of CD27L protein in the sample.The sample may be a blood sample or biopsy.

In a thirteenth aspect, the invention provides methods of treating anautoimmune or inflammatory disorder said method comprising administeringa therapeutically effective amount of a CD27L antigen binding protein ofany one of the first, second, third, fourth, fifth, or sixth aspects toa patient in need thereof. In preferred embodiments, the CD27L antigenbinding protein is an antibody comprising a light chain variable domainamino acid sequence as set forth in SEQ ID NO:63 and a heavy chainvariable domain amino acid sequence as set forth in SEQ ID NO:17 (e.g.,Ab1), an antibody comprising a light chain variable domain amino acidsequence as set forth in SEQ ID NO:64 and a heavy chain variable domainamino acid sequence as set forth in SEQ ID NO:18 (e.g., Ab2), anantibody comprising a light chain variable domain amino acid sequence asset forth in SEQ ID NO:65 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:19 (e.g., Ab3), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:66 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:20 (e.g., Ab4), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:67 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:21 (e.g., Ab5), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:68 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:22 (e.g., Ab6), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:69 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:23 (e.g., Ab7), or an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:70 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:24 (e.g., Ab8). In preferredembodiments, the CD27L antigen binding protein inhibits binding of CD27to CD27L. In particularly preferred embodiments, the autoimmune orinflammatory disorder is systemic lupus erythematosus (SLE), insulindependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD),multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoidarthritis (RA), or glomerulonephritis. In other embodiments, treatmentinhibits or prevents transplant rejection or graft versus host diseasein the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Summary of functional and physical characteristics of exemplaryembodiments of CD27L antigen binding proteins.

FIG. 2. Measurement of the level and the rate of internalization of Ab4(A4) and Ab8 (A8) and their conjugated counterparts into 786-0 cellsover time (A).

FIG. 3. 786-0 cells were cultured for 4 days in the presence ofincreasing concentrations of anti streptavidin-MCC-DM1, Ab4-MCC-DM1 andAb8-MCC-DM1. Effect on cell growth was measured using the CELL-TITER-GLOreagent that measures cell number using a luminescent read-out.

FIG. 4. Dose response of Ab4-MCC-DM1 and Ab8-MCC-DM1 in the 786-0xenograft model. Intravenous dose on day 24 is denoted by arrow at theindicated doses.

FIG. 5. Dose response of Ab4-MCC-DM1 and Ab8-MCC-DM1 in the Caki-1xenograft model. Intravenous dosing schedule is denoted by the arrows.

FIG. 6. Dose response of Ab4-MCC-DM1 in the Raji xenograft model. Theintravenous dosing schedule is denoted by the arrows.

FIG. 7. Variable dose response of Ab4-MCC-DM1 in the 786-0 xenograftmodel.

FIG. 8. Ab4-MCC-DM1 Antibody Dependent Cell Cytotoxicity (ADCC) Assayagainst Raji tumor cells.

FIG. 9. Ab4-MCC-DM1 Antibody Dependent Cellular Phagocytosis (ADCP)Assay against both Raji and 786-0 tumor cells.

FIG. 10. Comparison of CD27L expression for primary frozen tumor samplesthat scored “positive” by masked IHC. The results indicate that overallCD27L protein expression is highest in ccRCC patient samples, followedby DLBCL (Diffuse Large B-Cell Lymphoma); B-CLL and FL samples.

FIG. 11. Results of CD27L mRNA and Protein Expression Analysis. Theresults indicate that there is a high prevalence of CD27L expression inRCC, B-NHL and CLL cells.

FIG. 12. Sets forth the structure of the Ab-MCC-DM1 ADCs described inExample 2 herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited within the body of this specification are expresslyincorporated by reference in their entirety.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification, etc.Enzymatic reactions and purification techniques may be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manuel, 3^(rd) ed., Cold Spring Harbor Laboratory Press, coldSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,analytic chemistry, organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, chemicalanalyses, pharmaceutical preparation, formulation, and delivery andtreatment of patients.

CD27L

The antigen binding proteins bind to CD27L, which is also known as CD70and TNFSF7. CD27L was first described in U.S. Pat. No. 5,573,924. Anexemplary human CD27L amino acid sequence is provided herein as SEQ IDNO:1, which corresponds to NCBI Reference Sequence NP_001423.1(GI:4507605). In certain embodiments, the antigen binding protein blocksthe interaction of CD27L with its receptor CD27. An exemplary CD27 aminoacid sequence is provided as SEQ ID NO:2, which corresponds toSwiss-Prot: P26842.2 (GI:269849546). A mature CD27 amino acid sequencecorresponds to amino acids 20-260 of SEQ ID NO:2.

CD27L Antigen Binding Proteins

The present invention provides antigen binding proteins thatspecifically bind CD27L. Embodiments of antigen binding proteinscomprise peptides and/or polypeptides that specifically bind CD27L. Suchpeptides or polypeptides may optionally include one or moreport-translational modification. Embodiments of antigen binding proteinscomprise antibodies and fragments thereof, as variously defined herein,that specifically bind CD27L. These include antibodies that specificallybind human CD27L, including those that inhibit CD27L from binding and/oractivating CD27.

The antigen binding proteins of the invention specifically bind toCD27L. “Specifically binds” as used herein means that the antigenbinding protein preferentially binds CD27L over other proteins. In someembodiments “specifically binds” means the CD27L antigen binding proteinhas a higher affinity for CD27L than for other proteins. CD27L antigenbinding proteins that specifically bind CD27L may have a bindingaffinity for human CD27L of less than or equal to 1×10⁻⁷ M, less than orequal to 2×10⁻⁷ M, less than or equal to 3×10⁻⁷ M, less than or equal to4×10⁻⁷ M, less than or equal to 5×10⁻⁷ M, less than or equal to 6×10⁻⁷M, less than or equal to 7×10⁻⁷ M, less than or equal to 8×10⁻⁷ M, lessthan or equal to 9×10⁻⁷ M, less than or equal to 1×10⁻⁸ M, less than orequal to 2×10⁻⁸ M, less than or equal to 3×10⁻⁸ M, less than or equal to4×10⁻⁸ M, less than or equal to 5×10⁻⁸ M, less than or equal to 6×10⁻⁸M, less than or equal to 7×10⁻⁸ M, less than or equal to 8×10⁻⁸ M, lessthan or equal to 9×10⁻⁸ M, less than or equal to 1×10⁻⁹ M, less than orequal to 2×10⁻⁹ M, less than or equal to 3×10⁻⁹ M, less than or equal to4×10⁻⁹ M, less than or equal to 5×10⁻⁹ M, less than or equal to 6×10⁻⁹M, less than or equal to 7×10⁻⁹ M, less than or equal to 8×10⁻⁹ M, lessthan or equal to 9×10⁻⁹ M, less than or equal to 1×10⁻¹⁰ M, less than orequal to 2×10⁻¹⁰ M, less than or equal to 3×10⁻¹⁰ M, less than or equalto 4×10⁻¹⁰ M, less than or equal to 5×10⁻¹⁰ M, less than or equal to6×10⁻¹⁰ M, less than or equal to 7×10⁻¹⁰ M, less than or equal to8×10⁻¹⁰ M, less than or equal to 9×10⁻¹⁰ M, less than or equal to1×10⁻¹¹ M, less than or equal to 2×10⁻¹¹ M, less than or equal to3×10⁻¹¹ M, less than or equal to 4×10⁻¹¹ M, less than or equal to5×10⁻¹¹ M, less than or equal to 6×10⁻¹¹ M, less than or equal to7×10⁻¹¹ M, less than or equal to 8×10⁻¹¹ M, less than or equal to9×10⁻¹¹ M, less than or equal to 1×10⁻¹² M, less than or equal to2×10⁻¹² M, less than or equal to 3×10⁻¹² M, less than or equal to4×10⁻¹² M, less than or equal to 5×10⁻¹² M, less than or equal to6×10⁻¹² M, less than or equal to 7×10⁻¹² M, less than or equal to8×10⁻¹² M, or less than or equal to 9×10⁻¹² M. Methods of measuring thebinding affinity of an antigen binding protein are well known in theart. Example 1 provides an exemplary method.

It is understood that when reference is made to the various embodimentsof the CD27L-binding antibodies herein, that it also encompassesCD27L-binding fragments thereof. A CD27L-binding fragment comprises anyof the antibody fragments or domains described herein that retains theability to specifically bind to CD27L. The CD27L-binding fragment may bein any of the scaffolds described herein.

In certain therapeutic embodiments, a CD27L antigen binding proteininhibits binding of CD27L to CD27 and/or inhibits one or more biologicalactivities associated with the binding of CD27L to CD27, e.g.,CD27-mediated signaling. Such antigen binding proteins are said to be“neutralizing.” In certain embodiments, the neutralizing CD27L antigenbinding protein specifically binds CD27L and inhibits binding of CD27Lto CD27 from anywhere between 10% to 100%, such as by at least about 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99% or more. For example, CD27L antigen bindingproteins may be tested for neutralizing ability by determining theability of the antigen binding protein to block binding of CD27-Fc toMP-1 cells (Slack et al, Int. Immunol. (1995) 7(7): 1087-1092)).

Embodiments of antigen binding proteins comprise a scaffold structure,as variously defined herein, with one or more complementaritydetermining regions (CDRs). Embodiments further include antigen bindingproteins comprising a scaffold structure with one or more antibodyvariable domains, either heavy or light. Embodiments include antibodiesthat comprise a light chain variable domain selected from the groupconsisting of Ab1 Light Chain Variable Domain (LCv), Ab2 LCv, Ab3 LCv,Ab4 LCv, Ab5 LCv, Ab6 LCv, Ab7 LCv, and Ab8 LCv (SEQ ID NO:63-70,respectively) and/or a heavy chain variable domain selected from thegroup consisting of Ab1 Heavy Chain Variable Domain (HCv), Ab2 HCv, Ab3HCv, Ab4 HCv, Ab5 HCv, Ab6 HCv, Ab7 HCv, and Ab8 HCv (SEQ ID NO:17-24,respectively), and fragments, derivatives, muteins, and variantsthereof.

An exemplary light chain comprising Ab1 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:56.

An exemplary light chain comprising Ab2 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:57.

An exemplary light chain comprising Ab4 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:58.

An exemplary light chain comprising Ab5 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:59.

An exemplary light chain comprising Ab6 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:60.

An exemplary light chain comprising Ab7 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:61.

An exemplary light chain comprising Ab8 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:62.

An exemplary heavy chain comprising Ab1 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:10.

An exemplary heavy chain comprising Ab2 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:11.

An exemplary heavy chain comprising Ab4 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:12.

An exemplary heavy chain comprising Ab5 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:13.

An exemplary heavy chain comprising Ab6 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:14.

An exemplary heavy chain comprising Ab7 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:15.

An exemplary heavy chain comprising Ab8 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:16.

Additional examples of scaffolds that are envisioned include:fibronectin, neocarzinostatin CBM4-2, lipocalins, T-cell receptor,protein-A domain (protein Z), Im9, TPR proteins, zinc finger domains,pVIII, avian pancreatic polypeptide, GCN4, WW domain Src homology domain3, PDZ domains, TEM-1 beta-lactamase, thioredoxin, staphylococcalnuclease, PHD-finger domains, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-D1,LDTI, MTI-II, scorpion toxins, insect defensin-A peptide, EETI-II,Min-23, CBD, PBP, cytochrome b-562, Ldl receptor domains,gamma-crystallin, ubiquitin, transferrin, and or C-type lectin-likedomains. Non-antibody scaffolds and their use as therapeutics arereviewed in Gebauer and Skerra, Curr. Opin. Chem. Biol., 13:245-255(2009) and Binz et al., Nat. Biotech., 23(10):1257-1268 (2005), whichare incorporated herein by reference in its entirety.

Aspects of the invention include antibodies comprising the followingvariable domains: Ab1 LCv/Ab1 HCv (SEQ ID NO:63/SEQ ID NO:17), Ab2LCv/Ab2 HCv (SEQ ID NO:64/SEQ ID NO:18), Ab3 LCv/Ab3 HCv (SEQ IDNO:65/SEQ ID NO:19), Ab4 LCv/Ab4 HCv (SEQ ID NO:66/SEQ ID NO:20), Ab5LCv/Ab5 HCv (SEQ ID NO:67/SEQ ID NO:21), Ab6 LCv/Ab6 HCv (SEQ IDNO:68/SEQ ID NO:22), Ab7 LCv/Ab7 HCv (SEQ ID NO:69/SEQ ID NO:23), Ab8LCv/Ab8 HCv (SEQ ID NO:70/SEQ ID NO:24), and combinations thereof, aswell as fragments, derivatives, muteins and variants thereof.

Exemplary antibodies of the invention include Ab1 (SEQ ID NO:56/SEQ IDNO:10), Ab2 (SEQ ID NO:57/SEQ ID NO:11), Ab4 (SEQ ID NO:58/SEQ IDNO:12), Ab5 (SEQ ID NO:59/SEQ ID NO:13), Ab6 (SEQ ID NO:60/SEQ IDNO:14), Ab7 (SEQ ID NO:61/SEQ ID NO:15), Ab8 (SEQ ID NO:62/SEQ IDNO:16).

Typically, each variable domain of an antibody light or heavy chaincomprises three CDRs. The heavy chain variable domain comprises a heavychain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3(HCDR3). The light chain variable domain comprises a light chain CDR1(LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). Incertain embodiments, an antigen binding protein comprises one or moreCDRs contained within the preferred variable domains described herein.

Examples of such CDRs include, but are not limited to:

the CDRs of Ab1 LCv: LCDR1 (SEQ ID NO:71), LCDR2 (SEQ ID NO:79), andLCDR3 (SEQ ID NO:87);

the CDRs of Ab2 LCv: LCDR1 (SEQ ID NO:72), LCDR2 (SEQ ID NO:80), andLCDR3 (SEQ ID NO:88);

the CDRs of Ab3 LCv: LCDR1 (SEQ ID NO:73), LCDR2 (SEQ ID NO:81), andLCDR3 (SEQ ID NO:89);

the CDRs of Ab4 LCv: LCDR1 (SEQ ID NO:74), LCDR2 (SEQ ID NO:82), andLCDR3 (SEQ ID NO:90);

the CDRs of Ab5 LCv: LCDR1 (SEQ ID NO:75), LCDR2 (SEQ ID NO:83), andLCDR3 (SEQ ID NO:91);

the CDRs of Ab6 LCv: LCDR1 (SEQ ID NO:76), LCDR2 (SEQ ID NO:84), andLCDR3 (SEQ ID NO:92);

the CDRs of Ab7 LCv: LCDR1 (SEQ ID NO:77), LCDR2 (SEQ ID NO:85), andLCDR3 (SEQ ID NO:93);

the CDRs of Ab8 LCv: LCDR1 (SEQ ID NO:78), LCDR2 (SEQ ID NO:86), andLCDR3 (SEQ ID NO:94);

the CDRs of Ab1 HCv: HCDR1 (SEQ ID NO:25), HCDR2 (SEQ ID NO:33), andHCDR3 (SEQ ID NO:41);

the CDRs of Ab2 HCv: HCDR1 (SEQ ID NO:26), HCDR2 (SEQ ID NO:34), andHCDR3 (SEQ ID NO:42);

the CDRs of Ab3 HCv: HCDR1 (SEQ ID NO:27), HCDR2 (SEQ ID NO:35), andHCDR3 (SEQ ID NO:43);

the CDRs of Ab4 HCv: HCDR1 (SEQ ID NO:28), HCDR2 (SEQ ID NO:36), andHCDR3 (SEQ ID NO:44);

the CDRs of Ab5 HCv: HCDR1 (SEQ ID NO:29), HCDR2 (SEQ ID NO:37), andHCDR3 (SEQ ID NO:45);

the CDRs of Ab6 HCv: HCDR1 (SEQ ID NO:30), HCDR2 (SEQ ID NO:38), andHCDR3 (SEQ ID NO:46);

the CDRs of Ab7 HCv: HCDR1 (SEQ ID NO:31), HCDR2 (SEQ ID NO:39), andHCDR3 (SEQ ID NO:47); and

the CDRs of Ab8 HCv: HCDR1 (SEQ ID NO:32), HCDR2 (SEQ ID NO:40), andHCDR3 (SEQ ID NO:48).

In some embodiments, the antigen binding protein comprises: A) apolypeptide, e.g., a light chain, that comprises an LCDR1 having anamino acid sequence selected from the group consisting of SEQ ID NOS:71,72, 73, 74, 75, 76, 77, and 78; an LCDR2 having an amino acid sequenceselected from the group consisting of SEQ ID NOS: 79, 80, 81, 82, 83,84, 85, and 86; and/or an LCDR3 having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:87, 88, 89, 90, 91, 92, 93, and94; and/or B) a polypeptide, e.g., a heavy chain, that comprises anHCDR1 having an amino acid sequence selected from the group consistingof SEQ ID NOS:25, 26, 27, 28, 29, 30, 31, and 32; an HCDR2 having anamino acid sequence selected from the group consisting of SEQ ID NOS:33,34, 35, 36, 37, 38, 39, and 40; and/or an HCDR3 having an amino acidsequence selected from the group consisting of SEQ ID NOS:41, 42, 43,44, 45, 46, 47, and 48.

In further embodiments, the antigen binding protein comprise A) a lightchain amino acid sequence that comprises a LCDR1, LCDR2, and LCDR3 ofany of Ab1 LCv, Ab2 LCv, Ab3 LCv, Ab4 LCv, Ab5 LCv, Ab6 LCv, Ab7 LCv,and Ab8 LCv, and B) a heavy chain amino acid sequence that comprises aHCDR1, HCDR2, and HCDR3 of any of Ab1 HCv, Ab2 HCv, Ab3 HCv, Ab4 HCv,Ab5 HCv, Ab6 HCv, Ab7 HCv, and Ab8 HCv.

In certain embodiments, the CDRs include no more than one, no more thantwo, no more than three, no more than four, no more than five, or nomore than six amino acid additions, deletions, or substitutions from anexemplary CDR set forth herein.

Aspects of the invention include antibodies comprising a light chainvariable domain selected from the group consisting of SEQ ID NOS:63, 64,65, 66, 67, 68, 69, and 70. Aspects of the invention include antibodiescomprising a heavy chain variable domain selected from the groupconsisting of SEQ ID NOS:17, 18, 19, 20, 21, 22, 23, and 24. Furtheraspects of the invention include antibodies comprising A) a light chainvariable domain selected from the group consisting of SEQ ID NOS:63, 64,65, 66, 67, 68, 69, and 70, and B) a heavy chain variable domainselected from the group consisting of SEQ ID NOS:17, 18, 19, 20, 21, 22,23, and 24.

Antibodies of the invention can comprise any constant region known inthe art. The light chain constant region can be, for example, a kappa-or lambda-type light chain constant region, e.g., a human kappa- orlambda-type light chain constant region. The heavy chain constant regioncan be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-typeheavy chain constant region, e.g., a human alpha-, delta-, epsilon-,gamma-, or mu-type heavy chain constant region. In one embodiment thelight or heavy chain constant region is a fragment, derivative, variant,or mutein of a naturally occurring constant region.

Aspects of the invention include antibodies comprising a light chainvariable region selected from the group consisting of SEQ ID NOS:63, 64,65, 66, 67, 68, 69, and 70 having no more than one, no more than two, nomore than three, no more than four, no more than five, no more than six,no more than seven, no more than eight, no more than nine, or no morethan ten amino acid additions, deletions, or substitutions. Aspects ofthe invention include antibodies comprising a heavy chain variableregion selected from the group consisting of SEQ ID NOS:17, 18, 19, 20,21, 22, 23, and 24 having no more than one, no more than two, no morethan three, no more than four, no more than five, no more than six, nomore than seven, no more than eight, no more than nine, or no more thanten amino acid additions, deletions, or substitutions. Further aspectsof the invention include antibodies comprising A) comprising a lightchain variable region selected from the group consisting of SEQ IDNOS:63, 64, 65, 66, 67, 68, 69, and 70 having no more than one, no morethan two, no more than three, no more than four, no more than five, nomore than six, no more than seven, no more than eight, no more thannine, or no more than ten amino acid additions, deletions, orsubstitutions, and B) a heavy chain variable region selected from thegroup consisting of SEQ ID NOS:17, 18, 19, 20, 21, 22, 23, and 24 havingno more than one, no more than two, no more than three, no more thanfour, no more than five, no more than six, no more than seven, no morethan eight, no more than nine, or no more than ten amino acid additions,deletions, or substitutions. For example, in certain exemplaryembodiments, an antibody comprises 1) a variant of the light chainvariable domain set forth in SEQ ID NO:66, wherein the phenylalanine atposition 51 is mutated to a leucine and/or the proline at position 105is mutated to a glycine or a glutamine; 2) a variant of the heavy chainvariable domain set forth in SEQ ID NO:20, wherein the glutamine atposition 1 is mutated to glutamic acid and/or the arginine at position16 is mutated to a glycine; or a variant of the light chain variabledomain set forth in SEQ ID NO:66, wherein the phenylalanine at position51 is mutated to a leucine and/or the proline at position 105 is mutatedto a glycine or a glutamine and a variant of the heavy chain variabledomain set forth in SEQ ID NO:20, wherein the glutamine at position 1 ismutated to glutamic acid and/or the arginine at position 16 is mutatedto a glycine.

In one variation, the antigen binding protein comprises an amino acidsequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a light chain amino acid sequence selected fromthe group consisting of SEQ ID NOS:63, 64, 65, 66, 67, 68, 69, and 70.In another variation, the antigen binding protein comprises an aminoacid sequence that is at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NOS:17, 18, 19, 20, 21, 22,23, and 24. In yet a further embodiment, the antigen binding proteincomprises A) an amino acid sequence that is at least 80%, at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to a light chainamino acid sequence selected from the group consisting of SEQ ID NOS:63,64, 65, 66, 67, 68, 69, and 70, and B) an amino acid sequence that is atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NOS:17, 18, 19, 20, 21, 22, 23, and 24.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR3. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:87, 88, 89, 90, 91, 92, 93, 94, 41,42, 43, 44, 45, 46, 47, and 48. In certain embodiments, the amino acidsequence includes no more than one, no more than two, no more thanthree, no more than four, no more than five, or no more than six aminoacid additions, deletions, or substitutions from the exemplary sequenceset forth in SEQ ID NOS:87, 88, 89, 90, 91, 92, 93, 94, 41, 42, 43, 44,45, 46, 47, and 48. Thus, embodiments of the invention include antigenbinding protein comprising an amino acid sequence that is at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence selected from the group of sequences set forth inSEQ ID NOS:87, 88, 89, 90, 91, 92, 93, 94, 41, 42, 43, 44, 45, 46, 47,and 48.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR2. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:79, 80, 81, 82, 83, 84, 85, 86, 33,34, 35, 36, 37, 38, 39, and 40. In certain embodiments, the amino acidsequence includes no more than one, no more than two, no more thanthree, no more than four, no more than five, or no more than six aminoacid additions, deletions, or substitutions from the exemplary sequenceset forth in SEQ ID NOS: 79, 80, 81, 82, 83, 84, 85, 86, 33, 34, 35, 36,37, 38, 39, and 40. Thus, embodiments of the invention include antigenbinding protein comprising an amino acid sequence that is at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence selected from the group of sequences set forth inSEQ ID NOS: 79, 80, 81, 82, 83, 84, 85, 86, 33, 34, 35, 36, 37, 38, 39,and 40.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR1. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:71, 72, 73, 74, 75, 76, 77, 78, 25,26, 27, 28, 29, 30, 31, and 32. In certain embodiments, the amino acidsequence includes no more than one, no more than two, no more thanthree, no more than four, no more than five, or no more than six aminoacid additions, deletions, or substitutions from the exemplary sequenceset forth in SEQ ID NOS: 71, 72, 73, 74, 75, 76, 77, 78, 25, 26, 27, 28,29, 30, 31, and 32. Thus, embodiments of the invention include antigenbinding protein comprising an amino acid sequence that is at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence selected from the group of sequences set forth inSEQ ID NOS: 71, 72, 73, 74, 75, 76, 77, 78, 25, 26, 27, 28, 29, 30, 31,and 32.

The antigen binding proteins of the invention comprise the scaffolds oftraditional antibodies, including human and monoclonal antibodies,bispecific antibodies, diabodies, minibodies, domain antibodies,synthetic antibodies (sometimes referred to herein as “antibodymimetics”), chimeric antibodies, antibody fusions (sometimes referred toas “antibody conjugates”), and fragments of each, respectively. Theabove described CDRs, including various combinations of the CDRs, may begrafted into any of the following scaffolds.

As used herein, the term “antibody” refers to the various forms ofmonomeric or multimeric proteins comprising one or more polypeptidechains that specifically binds to an antigen, as variously describedherein. In certain embodiments, antibodies are produced by recombinantDNA techniques. In additional embodiments, antibodies are produced byenzymatic or chemical cleavage of naturally occurring antibodies. Inanother aspect, the antibody is selected from the group consisting of:a) a human antibody; b) a humanized antibody; c) a chimeric antibody; d)a monoclonal antibody; e) a polyclonal antibody; f) a recombinantantibody; g) an antigen-binding fragment; h) a single chain antibody; i)a diabody; j) a triabody, k) a tetrabody, 1) a Fab fragment; m) aF(ab′)₂ fragment, n) an IgA antibody, o) an IgD antibody, p) an IgEantibody, q) an IgG1 antibody, r) an IgG2 antibody, s) an IgG3 antibody,t) an IgG4 antibody, and u) an IgM antibody.

A variable region comprises at least three heavy or light chain CDRsembedded within a framework region (designated framework regions FR1,FR2, FR3, and FR4). Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, Public Health Service N.I.H., Bethesda, Md.Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” and one “heavy” chain.The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subclasses, including, but not limited to IgG1, IgG2, IgG3, andIgG4. IgM has subclasses, including, but not limited to IgM1 and IgM2.Embodiments of the invention include all such classes and subclasses ofantibodies that incorporate a variable domain or CDR of the antigenbinding proteins, as described herein.

Some naturally occurring antibodies, such as those found in camels andllamas, are dimers consisting of two heavy chains and include no lightchains. The invention encompasses dimeric antibodies of two heavychains, or fragments thereof, that can bind to CD27L.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three hypervariable regions, i.e., the complementaritydetermining regions or CDRs. The CDRs are primarily responsible forantigen recognition and binding. The CDRs fromt eh two chains of eachpair are aligned by the framework regions, enabling binding to aspecific epitope. From N-terminal to C-terminal, both light and heavychains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.The assignment of amino acids to each domain is in accordance with thedefinitions of Kabat.

CDRs constitute the major surface contact points for antigen binding.The CDR3 or the light chain and, particularly, CDR3 of the heavy chainmay constitute the most important determinants in antigen binding withinthe light and heavy chain variable regions. In some antibodies, theheavy chain CDR3 appears to constitute the major area of contact betweenthe antigen and the antibody. In vitro selection schemes in which CDR3alone is varied can be used to vary the binding properties of anantibody or determine which residues contribute to the binding of anantigen.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is typically not present in the mature antibody. Apolynucleotide encoding an antibody of the invention may encode anaturally occurring a signal sequence or a heterologous signal sequenceas described below.

In one embodiment, the antigen binding protein is a antibody comprisingfrom one to six of the exemplary CDRs described herein. The antibodiesof the invention may be of any type including IgM, IgG (including IgG1,IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. In a specific embodimentthe antigen biding protein is an IgG type antibody, e.g., a IgG1antibody.

In some embodiments, for example when the antigen binding protein is anantibody with complete heavy and light chains, the CDRs are all from thesame species, e.g., human. Alternatively, for example in embodimentswherein the antigen binding protein contains less than six CDRs from thesequences outlined above, additional CDRs may be either from otherspecies or may be different human CDRs than those depicted in theexemplary sequences. For example, HCDR3 and LCDR3 regions from theappropriate sequences identified herein may be used with HCDR1, HCDR2,LCDR1, and LCDR2 being optionally selected from alternate species ordifferent human antibody sequences, or combinations thereof. Forexample, the CDRs of the invention can replace the CDR regions ofcommercially relevant chimeric or humanized antibodies.

Specific embodiments utilize scaffold components of the antigen bindingproteins that are human components. In some embodiments, however, thescaffold components can be a mixture from different species. As such, ifthe antigen binding protein is an antibody, such antibody may be achimeric antibody and/or humanized antibody. In general, both “chimericantibodies” and humanized antibodies” refer to antibodies that combineregions from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse (or rat, in somecasaes) and the constant region(s) from a human.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except one or more CDRs, is encoded by a polynucleotide ofhuman origin or is identical to such an antibody except within one ormore CDRs. The CDRs, some or all of which are encoded by nucleic acidsoriginating in a non-human organism, are grafted into the beta-sheetframework of a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones 1986,Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. In the exemplary embodiments described herein, theidentified CDRs are human, and thus both humanized and chimericantibodies in this context include some non-human CDRs; for example,humanized antibodies may be generated that comprise the HCDR3 and LCDR3regions, with one or more of the other CDR regions being of a differentspecies origin.

In one embodiment, the CD27L antigen binding protein is a mutlispecificantibody, and notably a bispecfic antibody, also sometimes referred toas “diabodies.” These are antibodies that bind to two or more differentantigens or different epitopes on a single antigen. In certainembodiments, a bispecific antibody binds CD27L and an antigen on a humaneffector cell (e.g., T cell). Such antibodies are useful in targeting aneffector cell response against a CD27L expressing cells, such as a tumorcell. In preferred embodiments, the human effector cell antigen is CD3.U.S. Pat. No. 7,235,641. Methods of making bispecific antibodies areknown in the art. One such method involves engineering the Fc portion ofthe heavy chains such as to create “knobs” and “holes” which facilitateheterodimer formation of the heavy chains when co-expressed in a cell.U.S. Pat. No. 7,695,963. Another method also involves engineering the Fcportion of the heavy chain but uses electrostatic steering to encourageheterodimer formation while discouraging homodimer formation of theheavy chains when co-expressed in a cell. WO 09/089,004, which isincorporated herein by reference in its entirety.

In one embodiment, the CD27L antigen binding protein is a minibody.Minibodies are minimized antibody-like proteins comprising a scFv joinedto a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061.

In one embodiment, the CD27L antigen binding protein is a domainantibody; see, for example U.S. Pat. No. 6,248,516. Domain antibodies(dAbs) are functional binding domains of antibodies, corresponding tothe variable regions of either the heavy (VH) or light (VL) chains ofhuman antibodies. dABs have a molecular weight of approximately 13 kDa,or less than one-tenth the size of a full antibody. dABs are wellexpressed in a variety of hosts including bacterial, yeast, andmammalian cell systems. In addition, dAbs are highly stable and retainactivity even after being subjected to harsh conditions, such asfreeze-drying or heat denaturation. See, for example, U.S. Pat. Nos.6,291,158; 6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941;European Patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821,WO04/003019 and WO03/002609.

In one embodiment, the CD27L antigen binding protein is an antibodyfragment, that is a fragment of any of the antibodies outlined hereinthat retain binding specificity to CD27L. In various embodiments, theantibody binding proteins comprise, but are not limited to, a F(ab),F(ab′), F(ab′)2, Fv, or a single chain Fv fragments. At a minimum, anantibody, as meant herein, comprises a polypeptide that can bindspecifically to CD27L comprising all or part of a light or heavy chainvariable region, such as one or more CDRs.

Further examples of CD27L-binding antibody fragments include, but arenot limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1domains, (ii) the Fd fragment consisting of the VH and CH1 domains,(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546)which consists of a single variable, (v) isolated CDR regions, (vi)F(ab′)₂ fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988,Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:5879-5883), (viii) bispecific single chain Fv dimers (PCT/US92/09965)and (ix) “diabodies” or “triabodies”, multivalent or multispecificfragments constructed by gene fusion (Tomlinson et. al., 2000, MethodsEnzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl.Acad. Sci. U.S.A. 90:6444-6448). The antibody fragments may be modified.For example, the molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al., 1996,Nature Biotech. 14:1239-1245). Aspects of the invention includeembodiments wherein the non-CDR components of these fragments are humansequences.

In one embodiment, the CD27L antigen binding protein is a fully humanantibody. In this embodiment, as outlined above, specific structurescomprise complete heavy and light chains depicted comprising the CDRregions. Additional embodiments utilize one or more of the CDRs of theinvention, with the other CDRs, framework regions, J and D regions,constant regions, etc., coming from other human antibodies. For example,the CDRs of the invention can replace the CDRs of any number of humanantibodies, particularly commercially relevant antibodies

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol Biol. 178:379-87. Single chain antibodies derivedfrom antibodies provided herein (including but not limited to scFvscomprising the variable domain combinations of Ab1 LCv/Ab1 HCv (SEQ IDNO:63/SEQ ID NO:17), Ab2 LCv/Ab2 HCv (SEQ ID NO:64/SEQ ID NO:18), Ab3LCv/Ab3 HCv (SEQ ID NO:65/SEQ ID NO:19), Ab4 LCv/Ab4 HCv (SEQ IDNO:66/SEQ ID NO:20), Ab5 LCv/Ab5 HCv (SEQ ID NO:67/SEQ ID NO:21), Ab6LCv/Ab6 HCv (SEQ ID NO:68/SEQ ID NO:22), Ab7 LCv/Ab7 HCv (SEQ IDNO:69/SEQ ID NO:23), Ab8 LCv/Ab8 HCv (SEQ ID NO:70/SEQ ID NO:24), andcombinations thereof are encompassed by the present invention.

In one embodiment, the CD27L antigen binding protein is an antibodyfusion protein (sometimes referred to herein as an “antibodyconjugate”). The conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antigen binding protein and on the conjugate partner. Incertain embodiments, the antibody is conjugated to a non-proteinaceouschemical (drug) to form an antibody drug conjugate. Exemplary antibodydrug conjugates and methods of making such conjugates are discussedbelow.

In one embodiment, the CD27L antigen binding protein is an antibodyanalog, sometimes referred to as “synthetic antibodies.” For example, avariety of work utilizes either alternative protein scaffolds orartificial scaffolds with grafted CDRs. Such scaffolds include, but arenot limited to, mutations introduced to stabilize the three-dimensionalstructure of the binding protein as well as wholly synthetic scaffoldsconsisting for example of biocompatible polymers. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as work based on antibody mimeticsutilizing fibronection components as a scaffold.

By “protein,” as used herein, is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells,as outlined below. Alternatively, the protein may include syntheticamino acids (e.g., homophenylalanine, citrulline, ornithine, andnorleucine), or peptidomimetic structures, i.e., “peptide or proteinanalogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad.Sci. U.S.A. 89:9367, incorporated by reference herein), which can beresistant to proteases or other physiological and/or storage conditions.Such synthetic amino acids may be incorporated in particular when theantigen binding protein is synthesized in vitro by conventional methodswell known in the art. In addition, any combination of peptidomimetic,synthetic and naturally occurring residues/structures can be used.“Amino acid” also includes imino acid residues such as proline andhydroxyproline. The amino acid “R group” or “side chain” may be ineither the (L)- or the (S)-configuration. In a specific embodiment, theamino acids are in the (L)- or (S)-configuration.

In certain aspects, the invention provides recombinant antigen bindingproteins that bind a CD27L and, in some embodiments, a recombinant humanCD27L or portion thereof. In this context, a “recombinant protein” is aprotein made using recombinant techniques using any techniques andmethods known in the art, i.e., through the expression of a recombinantnucleic acid as described herein. Methods and techniques for theproduction of recombinant proteins are well known in the art.Embodiments of the invention include recombinant antigen bindingproteins that bind wild-type CD27L and variants thereof.

“Consisting essentially of” means that the amino acid sequence can varyby about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% relativeto the recited SEQ ID NO: sequence and still retain biological activity,as described herein.

In some embodiments, the antigen binding proteins of the invention areisolated proteins or substantially pure proteins. An “isolated” proteinis unaccompanied by at least some of the material with which it isnormally associated in its natural state, for example constituting atleast about 5%, or at least about 50% by weight of the total protein ina given sample. It is understood that the isolated protein mayconstitute from 5 to 99.9% by weight of the total protein contentdepending on the circumstances. For example, the protein may be made ata significantly higher concentration through the use of an induciblepromoter or high expression promoter, such that the protein is made atincreased concentration levels. The definition includes the productionof an antigen binding protein in a wide variety of organisms and/or hostcells that are known in the art.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs are at least 80% to the sequences depictedherein, and more typically with preferably increasing homologies oridentities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, and almost 100%. In a similar manner, “percent (%) nucleic acidsequence identity” with respect to the nucleic acid sequence of thebinding proteins identified herein is defined as the percentage ofnucleotide residues in a candidate sequence that are identical with thenucleotide residues in the coding sequence of the antigen bindingprotein. A specific method utilizes the BLASTN module of WU-BLAST-2 setto the default parameters, with overlap span and overlap fraction set to1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andthe nucleotide sequences depicted herein are at least 80%, and moretypically with preferably increasing homologies or identities of atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.

Thus, a “variant CDR” is one with the specified homology, similarity, oridentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the specificity and/or activity of the parent CDR.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of antigen binding protein activities, such as CD27L binding.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions may betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as TABLE 1.

TABLE 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTABLE 1. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the antigen binding protein proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the antigen binding protein is altered. For example,glycosylation sites may be altered or removed as discussed herein.

Other derivatives of CD27L antibodies within the scope of this inventioninclude covalent or aggregative conjugates of CD27L antibodies, orfragments thereof, with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of a CD27L antibodypolypeptide. For example, the conjugated peptide may be a heterologoussignal (or leader) polypeptide, e.g., the yeast alpha-factor leader, ora peptide such as an epitope tag. CD27L antibody-containing fusionproteins can comprise peptides added to facilitate purification oridentification of the CD27L antibody (e.g., poly-His). A CD27L antibodypolypeptide also can be linked to the FLAG peptide as described in Hoppet al., Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912. TheFLAG peptide is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody (mAb), enabling rapid assay andfacile purification of expressed recombinant protein. Reagents usefulfor preparing fusion proteins in which the FLAG peptide is fused to agiven polypeptide are commercially available (Sigma, St. Louis, Mo.).

Oligomers that contain one or more CD27L antibody polypeptides may beemployed as CD27L antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more CD27L antibody polypeptidesare contemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple CD27Lantibody polypeptides joined via covalent or non-covalent interactionsbetween peptide moieties fused to the CD27L antibody polypeptides. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of CD27L antibody polypeptides attached thereto,as described in more detail below.

In particular embodiments, the oligomers comprise from two to four CD27Lantibody polypeptides. The CD27L antibody moieties of the oligomer maybe in any of the forms described above, e.g., variants or fragments.Preferably, the oligomers comprise CD27L antibody polypeptides that haveCD27L binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a CD27L bindingfragment of a CD27L antibody to the Fc region of an antibody. The dimercan be made by, for example, inserting a gene fusion encoding the fusionprotein into an appropriate expression vector, expressing the genefusion in host cells transformed with the recombinant expression vector,and allowing the expressed fusion protein to assemble much like antibodymolecules, whereupon interchain disulfide bonds form between the Fcmoieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of a CD27L antibody may be substituted for the variable portionof an antibody heavy and/or light chain.

Alternatively, the oligomer is a fusion protein comprising multipleCD27L antibody polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric CD27L antibody derivativesinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising CD27L antibody fragmentor derivative fused to a leucine zipper peptide are expressed insuitable host cells, and the soluble oligomeric CD27L antibody fragmentsor derivatives that form are recovered from the culture supernatant.

Covalent modifications of antigen binding proteins are included withinthe scope of this invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antigen binding protein are introduced into themolecule by reacting specific amino acid residues of the antigen bindingprotein with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantigen binding proteins to a water-insoluble support matrix or surfacefor use in a variety of methods. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the antigen binding proteinincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the target polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antigen binding proteincomprises linking the antigen binding protein to variousnonproteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, asis known in the art, amino acid substitutions may be made in variouspositions within the antigen binding protein to facilitate the additionof polymers such as PEG.

In some embodiments, the covalent modification of the antigen bindingproteins of the invention comprises the addition of one or more labels.

The term “labelling group” means any detectable label. Examples ofsuitable labelling groups include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labelling group is coupled to theantigen binding protein via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and may be used in performing the present invention.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabelling proteins are known in the art and may be used in performingthe present invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

Polynucleotides Encoding CD27L Antigen Binding Proteins

Encompassed within the invention are nucleic acids encoding CD27Lantigen binding proteins, including antibodies, as defined herein.Preferred nucleic acids include those that encode the exemplary lightand heavy chains described herein.

An exemplary nucleic acid encoding Ab1 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:49.

An exemplary nucleic acid encoding Ab2 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:50.

An exemplary nucleic acid encoding Ab4 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:51.

An exemplary nucleic acid encoding Ab5 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:52.

An exemplary nucleic acid encoding Ab6 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:53.

An exemplary nucleic acid encoding Ab7 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:54.

An exemplary nucleic acid encoding Ab8 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:55.

An exemplary nucleic acid encoding Ab1 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:3.

An exemplary nucleic acid encoding Ab2 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:4.

An exemplary nucleic acid encoding Ab4 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:5.

An exemplary nucleic acid encoding Ab5 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:6.

An exemplary nucleic acid encoding Ab6 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:7.

An exemplary nucleic acid encoding Ab7 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:8.

An exemplary nucleic acid encoding Ab8 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:9.

Aspects of the invention include polynucleotide variants (e.g., due todegeneracy) that encode the amino acid sequences described herein.

Aspects of the invention include a variety of embodiments including, butnot limited to, the following exemplary embodiments.

An isolated polynucleotide, wherein said polynucleotide encodes one ormore polypeptides comprising an amino acid sequence selected from thegroup consisting of:

A. 1. a light chain variable domain sequence that is at least 90%identical to a light chain variable domain sequence set forth in SEQ IDNOs:63-70;

-   -   2. a heavy chain variable domain sequence that is at least 90%        identical to a heavy chain variable domain sequence set forth in        SEQ ID NOs:17-24;    -   3. a light chain variable domain of (1) and a heavy chain        variable domain of (2); and

B. a light chain variable domain comprising a CDR1, CDR2, CDR3 and/or aheavy chain variable domain comprising a CDR1, CDR2, CDR3 that differ byno more than a total of three amino acid additions, substitutions,and/or deletions in each CDR from the following sequences:

-   -   1. a light chain CDR1 (SEQ ID NO:71), CDR2 (SEQ ID NO:79), CDR3        (SEQ ID NO:87) or a heavy chain CDR1 (SEQ ID NO:25), CDR2 (SEQ        ID NO:33), CDR3 (SEQ ID NO:41) of Ab1;    -   2. a light chain CDR1 (SEQ ID NO:72), CDR2 (SEQ ID NO:80), CDR3        (SEQ ID NO:88) or a heavy chain CDR1 (SEQ ID NO:26), CDR2 (SEQ        ID NO:34), CDR3 (SEQ ID NO:42) of Ab2;    -   3. a light chain CDR1 (SEQ ID NO:73), CDR2 (SEQ ID NO:81), CDR3        (SEQ ID NO:89) or a heavy chain CDR1 (SEQ ID NO:27), CDR2 (SEQ        ID NO:35), CDR3 (SEQ ID NO:43) of Ab3;    -   4. a light chain CDR1 (SEQ ID NO:74), CDR2 (SEQ ID NO:82), CDR3        (SEQ ID NO:90) or a heavy chain CDR1 (SEQ ID NO:28), CDR2 (SEQ        ID NO:36), CDR3 (SEQ ID NO:44) of Ab4;    -   5. a light chain CDR1 (SEQ ID NO:75), CDR2 (SEQ ID NO:83), CDR3        (SEQ ID NO:91) or a heavy chain CDR1 (SEQ ID NO:29), CDR2 (SEQ        ID NO:37), CDR3 (SEQ ID NO:45) of Ab5;    -   6. a light chain CDR1 (SEQ ID NO:76), CDR2 (SEQ ID NO:84), CDR3        (SEQ ID NO:92) or a heavy chain CDR1 (SEQ ID NO:30), CDR2 (SEQ        ID NO:38), CDR3 (SEQ ID NO:46) of Ab6;    -   7. a light chain CDR1 (SEQ ID NO:77), CDR2 (SEQ ID NO:85), CDR3        (SEQ ID NO:93) or a heavy chain CDR1 (SEQ ID NO:31), CDR2 (SEQ        ID NO:39), CDR3 (SEQ ID NO:47) of Ab7; and    -   8. a light chain CDR1 (SEQ ID NO:78), CDR2 (SEQ ID NO:86), CDR3        (SEQ ID NO:94) or a heavy chain CDR1 (SEQ ID NO:32), CDR2 (SEQ        ID NO:40), CDR3 (SEQ ID NO:48) of Ab8.

In preferred embodiments, the polypeptide encoded by the isolatednucleic acid is a component of an antigen binding protein that bindsCD27L.

Nucleotide sequences corresponding to the amino acid sequences describedherein, to be used as probes or primers for the isolation of nucleicacids or as query sequences for database searches, can be obtained by“back-translation” from the amino acid sequences, or by identificationof regions of amino acid identity with polypeptides for which the codingDNA sequence has been identified. The well-known polymerase chainreaction (PCR) procedure can be employed to isolate and amplify a DNAsequence encoding a CD27L antigen binding proteins or a desiredcombination of CD27L antigen binding protein polypeptide fragments.Oligonucleotides that define the desired termini of the combination ofDNA fragments are employed as 5′ and 3′ primers. The oligonucleotidescan additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified combination ofDNA fragments into an expression vector. PCR techniques are described inSaiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu etal., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCRProtocols: A Guide to Methods and Applications, Innis et. al., eds.,Academic Press, Inc. (1990).

Nucleic acid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. The nucleic acid molecules of the invention include full-lengthgenes or cDNA molecules as well as a combination of fragments thereof.The nucleic acids of the invention are preferentially derived from humansources, but the invention includes those derived from non-humanspecies, as well.

An “isolated nucleic acid” is a nucleic acid that has been separatedfrom adjacent genetic sequences present in the genome of the organismfrom which the nucleic acid was isolated, in the case of nucleic acidsisolated from naturally-occurring sources. In the case of nucleic acidssynthesized enzymatically from a template or chemically, such as PCRproducts, cDNA molecules, or oligonucleotides for example, it isunderstood that the nucleic acids resulting from such processes areisolated nucleic acids. An isolated nucleic acid molecule refers to anucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

The present invention also includes nucleic acids that hybridize undermoderately stringent conditions, and more preferably highly stringentconditions, to nucleic acids encoding CD27L antigen binding proteins asdescribed herein. The basic parameters affecting the choice ofhybridization conditions and guidance for devising suitable conditionsare set forth by Sambrook, Fritsch, and Maniatis (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11; and Current Protocols inMolecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4), and can be readily determined by thosehaving ordinary skill in the art based on, for example, the lengthand/or base composition of the DNA. One way of achieving moderatelystringent conditions involves the use of a prewashing solutioncontaining 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization bufferof about 50% formamide, 6×SSC, and a hybridization temperature of about55 degrees C. (or other similar hybridization solutions, such as onecontaining about 50% formamide, with a hybridization temperature ofabout 42 degrees C.), and washing conditions of about 60 degrees C., in0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined ashybridization conditions as above, but with washing at approximately 68degrees C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mMNaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC(1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization andwash buffers; washes are performed for 15 minutes after hybridization iscomplete. It should be understood that the wash temperature and washsalt concentration can be adjusted as necessary to achieve a desireddegree of stringency by applying the basic principles that governhybridization reactions and duplex stability, as known to those skilledin the art and described further below (see, e.g., Sambrook et al.,1989). When hybridizing a nucleic acid to a target nucleic acid ofunknown sequence, the hybrid length is assumed to be that of thehybridizing nucleic acid. When nucleic acids of known sequence arehybridized, the hybrid length can be determined by aligning thesequences of the nucleic acids and identifying the region or regions ofoptimal sequence complementarity. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be 5to 10.degrees C. less than the melting temperature (Tm) of the hybrid,where Tm is determined according to the following equations. For hybridsless than 18 base pairs in length, Tm (degrees C.)=2(# of A+T bases)+4(#of #G+C bases). For hybrids above 18 base pairs in length, Tm (degreesC.)=81.5+16.6(log₁₀ [Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165M). Preferably, each suchhybridizing nucleic acid has a length that is at least 15 nucleotides(or more preferably at least 18 nucleotides, or at least 20 nucleotides,or at least 25 nucleotides, or at least 30 nucleotides, or at least 40nucleotides, or most preferably at least 50 nucleotides), or at least25% (more preferably at least 50%, or at least 60%, or at least 70%, andmost preferably at least 80%) of the length of the nucleic acid of thepresent invention to which it hybridizes, and has at least 60% sequenceidentity (more preferably at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, and mostpreferably at least 99.5%) with the nucleic acid of the presentinvention to which it hybridizes, where sequence identity is determinedby comparing the sequences of the hybridizing nucleic acids when alignedso as to maximize overlap and identity while minimizing sequence gaps asdescribed in more detail above.

The variants according to the invention are ordinarily prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the antigenbinding protein, using cassette or PCR mutagenesis or other techniqueswell known in the art, to produce DNA encoding the variant, andthereafter expressing the recombinant DNA in cell culture as outlinedherein. However, antigen binding protein fragments comprising variantCDRs having up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, e.g., binding to CD27L, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the CDRs (and heavy and light chains or othercomponents of the antigen binding protein) of the present invention.Thus, having identified a particular amino acid sequence, those skilledin the art could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the encoded protein.

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the CD27Lantigen binding protein coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the CD27L antigen binding protein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified CD27L antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thyrnidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein antibody that binds to CD27L polypeptide. As aresult, increased quantities of a polypeptide such as an CD27L antigenbinding protein are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof rnRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed. In certain embodiments, one or more coding regions may beoperably linked to an internal ribosome binding site (IRES), allowingtranslation of two open reading frames from a single RNA transcript.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the CD27L antigen binding protein. Promoters areuntranscribed sequences located upstream (i.e., 5′) to the start codonof a structural gene (generally within about 100 to 1000 bp) thatcontrol transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an CD27L antigen binding protein of theinvention by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence into thevector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising aCD27L antigen binding protein of the invention by higher eukaryotes.Enhancers are cis-acting elements of DNA, usually about 10-300 bp inlength, that act on the promoter to increase transcription. Enhancersare relatively orientation and position independent, having been foundat positions both 5′ and 3′ to the transcription unit. Several enhancersequences available from mammalian genes are known (e.g., globin,elastase, albumin, alpha-feto-protein and insulin). Typically, however,an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The vector may contain one or more elements that facilitate expressionwhen the vector is integrated into the host cell genome. Examplesinclude an EASE element (Aldrich et al. 2003 Biotechnol Prog.19:1433-38) and a matrix attachment region (MAR). MARs mediatestructural organization of the chromatin and may insulate the integratedvactor from “position” effect. Thus, MARs are particularly useful whenthe vector is used to create stable transfectants. A number of naturaland synthetic MAR-containing nucleic acids are known in the art, e.g.,U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974;7,259,010; 6,037,525; 7,422,874; 7,129,062.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an CD27L antigen binding sequence has been inserted into theproper site of the vector, the completed vector may be inserted into asuitable host cell for amplification and/or polypeptide expression. Thetransformation of an expression vector for an CD27L antigen bindingprotein into a selected host cell may be accomplished by well knownmethods including transfection, infection, calcium phosphateco-precipitation, electroporation, microinjection, lipofection,DEAE-dextran mediated transfection, or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anCD27L antigen binding protein that can subsequently be collected fromthe culture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule. A host cell may be eukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC) and any celllines used in an expression system known in the art can be used to makethe recombinant polypeptides of the invention. In general, host cellsare transformed with a recombinant expression vector that comprises DNAencoding a desired anti-CD27L antibody polypeptide. Among the host cellsthat may be employed are prokaryotes, yeast or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include insect cellsand established cell lines of mammalian origin. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells,C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells,or their derivatives such as Veggie CHO and related cell lines whichgrow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cellline derived from the African green monkey kidney cell line CVI (ATCCCCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, humanembryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermalA431 cells, human Colo205 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49,for example, can be used for expression of the polypeptide when it isdesirable to use the polypeptide in various signal transduction orreporter assays. Alternatively, it is possible to produce thepolypeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Suitable yeasts include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous polypeptides. Suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous polypeptides. If the polypeptide is made in yeast orbacteria, it may be desirable to modify the polypeptide producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain the functional polypeptide. Suchcovalent attachments can be accomplished using known chemical orenzymatic methods. The polypeptide can also be produced by operablylinking the isolated nucleic acid of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987), andLuckow and Summers, Bio/Technology 6:47 (1988). Cell-free translationsystems could also be employed to produce polypeptides using RNAsderived from nucleic acid constructs disclosed herein. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, New York, 1985). A host cellthat comprises an isolated nucleic acid of the invention, preferablyoperably linked to at least one expression control sequence, is a“recombinant host cell”.

In certain embodiments, cell lines may be selected through determiningwhich cell lines have high expression levels and constitutively produceantigen binding proteins with CD27L binding properties. In anotherembodiment, a cell line from the B cell lineage that does not make itsown antibody but has a capacity to make and secrete a heterologousantibody can be selected.

Antibody Drug Conjugates

Embodiments of the invention include antibody drug conjugates (ADCs).Generally the ADC comprises an antibody conjugated to a chemotherapeuticagent, e.g., a cytotoxic agent, a cytostatic agent, a toxin, or aradioactive agent. A linker molecule can be used to conjugate the drugto the antibody. A wide variety of linkers and drugs useful in ADCtechnology are known in the art and may be used in embodiments of thepresent invention. (See US20090028856; US2009/0274713; US2007/0031402;WO2005/084390; WO2009/099728; U.S. Pat. No. 5,208,020; U.S. Pat. No.5,416,064; U.S. Pat. Nos. 5,475,092; 5,585,499; 6,436,931; 6,372,738;and 6,340,701, all incorporated herein by reference).

The antibody drug conjugates may be prepared by in vitro methods. Inorder to link a drug or prodrug to the antibody, a linking group isused. Suitable linking groups are well known in the art and includedisulfide groups, acid labile groups, photolabile groups, peptidaselabile groups, and esterase labile groups. Preferred linking groups aredisulfide groups. For example, conjugates can be constructed using adisulfide exchange reaction between the antibody and the drug orprodrug. The drug molecules also can be linked to a cell binding agentthrough an intermediary carrier molecule such as serum albumin.

In certain embodiments, the cell binding agent is modified by reacting abifunctional crosslinking reagent with the cell binding agent, therebyresulting in the covalent attachment of a linker molecule to the cellbinding agent. As used herein, a “bifunctional crosslinking reagent” or“linker” is any chemical moiety that covalently links a cell bindingagent to a drug, such as the drugs described herein. In a particularembodiment of the invention, a portion of the linking moiety is providedby the drug. In this respect, the drug comprises a linking moiety thatis part of a larger linker molecule that is used to join the cellbinding agent to the drug. For example, to form the maytansinoid DM1,the side chain at the C-3 hydroxyl group of maytansine is modified tohave a free sulfhydryl group (SH). This thiolated form of maytansine canreact with a modified cell-binding agent to form a conjugate. Therefore,the final linker is assembled from two components, one of which isprovided by the crosslinking reagent, while the other is provided by theside chain from DM1.

Any suitable bifunctional crosslinking reagent can be used in connectionwith the invention, so long as the linker reagent provides for retentionof the therapeutic, e.g., cytotoxicity, and targeting characteristics ofthe drug and the cell binding agent, respectively. Preferably, thelinker molecule joins the drug to the cell binding agent throughchemical bonds (as described above), such that the drug and the cellbinding agent are chemically coupled (e.g., covalently bonded) to eachother.

Linkers

In certain embodiments, the ADC comprises a linker made up of one ormore linker components. Preferably the drug is linked to a cell bindingagent through a disulfide bond. The linker molecule comprises a reactivechemical group that can react with the cell binding agent. Preferredreactive chemical groups for reaction with the cell binding agent areN-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally thelinker molecule comprises a reactive chemical group, preferably adithiopyridyl group, that can react with the drug to form a disulfidebond. Exemplary linker molecules include, for example, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) (see, e.g., Carlsson et al.,Biochem. J., 173, 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304)and N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see, e.g., CASRegistry number 341498-08-6). Additional exemplary linker componentsinclude 6-maleimidocaproyl, maleimidopropanoyl, valine-citrulline,alanine-phenylalanine, p-aminobenzyloxycarbonyl, and those resultingfrom conjugation with linker reagents, including, but not limited to,N-succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC,” also referredto herein also as “MCC”), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”).

Linkers may be a “cleavable” linker or a “non-cleavable” linker (Ducryand Stump, Bioconjugate Chem. 2010, 21, 5-13; incorporated herein byreference in its entirety) Cleavable linkers are designed to release thedrug when subjected to certain environment factors, e.g., wheninternalized into the target cell. Cleavable linkers include acid labilelinkers, protease sensitive linkers, photolabile linkers, dimethyllinker or disulfide-containing linkers. Non-cleavable linkers tend toremain covalently associated with at least one amino acid of theantibody and the drug upon internalization by and degradation within thetarget cell. A non-cleavable linker is any chemical moiety that iscapable of linking a drug, such as a maytansinoid (e.g., DM1, and thelike), a taxane, or a CC-1065 analog, to a cell binding agent in astable, covalent manner. Thus, non-cleavable linkers are substantiallyresistant to acid-induced cleavage, light-induced cleavage,peptidase-induced cleavage, esterase-induced cleavage, and disulfidebond cleavage, at conditions under which the drug or the cell bindingagent remains active.

Suitable crosslinking reagents that form non-cleavable linkers between adrug and the cell-binding agent are well known in the art. Examples ofnon-cleavable linkers include linkers having an N-succinimidyl ester orN-sulfosuccinimidyl ester moiety for reaction with the cell bindingagent, as well as a maleimido- or haloacetyl-based moiety for reactionwith the drug. Crosslinking linker reagents comprising a maleimido-basedmoiety include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproa-te),which is a “long chain” analog of SMCC (LC-SMCC), x-maleimidoundecanoicacid N-succinimidyl ester (KMUA), .gamma.-maleimidobutyric acidN-succinimidyl ester (GMBS), .epsilon.-maleimidocaproic acidN-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(.alpha.-maleimidoacetoxy)-succinimide ester (AMAS),succinimidyl-6-(beta.-maleimidopropionamido)hexanoate (SMPH),N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI). Cross-linking reagentscomprising a haloacetyl-based moiety includeN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl3-(bromoacetamido)propionate (SBAP). An exemplary preferrednon-cleavable linker is MCC.

Other crosslinking reagent linkers lacking a sulfur atom that formnon-cleavable linkers can also be used in the inventive method. Suchlinkers can be derived from dicarboxylic acid based moieties. Suitabledicarboxylic acid based moieties include, but are not limited to,alpha,omega-dicarboxylic acids of the general formula (IX):HOOC—X₁—Y_(n)—Z_(m)—COOH  (IX),wherein X is a linear or branched alkyl, alkenyl, or alkynyl grouphaving 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl groupbearing 3 to 10 carbon atoms, Z is a substituted or unsubstitutedaromatic group bearing 6 to 10 carbon atoms, or a substituted orunsubstituted heterocyclic group wherein the hetero atom is selectedfrom N, O or S, and wherein 1, m, and n are each 0 or 1, provided that1, m, and n are all not zero at the same time.

Many of the non-cleavable linkers disclosed herein are described indetail in U.S. Patent Application Publication 2005/0169933 A1.

Examples of suitable cleavable linkers include disulfide linkers, acidlabile linkers, photolabile linkers, peptidase labile linkers, andesterase labile linkers. Disulfide containing linkers are linkerscleavable through disulfide exchange, which can occur underphysiological conditions. Acid labile linkers are linkers cleavable atacid pH. For example, certain intracellular compartments, such asendosomes and lysosomes, have an acidic pH (pH 4-5), and provideconditions suitable to cleave acid labile linkers. Photo labile linkersare useful at the body surface and in many body cavities that areaccessible to light. Furthermore, infrared light can penetrate tissue.Peptidase labile linkers can be used to cleave certain peptides insideor outside cells (see e.g., Trouet et al., Proc. Natl. Acad. Sci. USA,79, 626-629 (1982), and Umemoto et al., Int. J. Cancer, 43, 677-684(1989)).Drugs

In certain embodiments, the antibody is conjugated to a chemotherapeuticagent. Examples of chemotherapeutic agents include alkylating agents,such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines, such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics, such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin .gamma1 and calicheamicin thetaI, see, e.g., Angew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites, such as methotrexate and5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens, such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals, such asaminoglutethimide, mitotane, trilostane; folic acid replenisher, such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids, such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;siRNA and pharmaceutically acceptable salts, acids or derivatives of anyof the above. Other chemotherapeutic agents that can be used with thepresent invention are disclosed in US Publication No. 20080171040 or USPublication No. 20080305044 and are incorporated in their entirety byreference.

It is contemplated that an antibody may be conjugated to two or moredifferent chemotherapeutic agents or a pharmaceutical composition maycomprise a mixture of antibodies wherein the antibody component isidentical except for being conjugated to a different chemotherapeuticagent. Such embodiments may be useful for targeting multiple biologicalpathways with a target cell.

In preferred embodiments, the ADC comprises an antibody conjugated toone or more maytansinoid molecules, which are mitotic inhibitors thatact by inhibiting tubulin polymerization. Maytansinoids, includingvarious modifications, are described in U.S. Pat. Nos. 3,896,111;4,151,042; 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; 4,371,533; and WO 2009/099728. Maytansinoid drugmoieties may be isolated from natural sources, produced usingrecombinant technology, or prepared synthetically. Exemplarymaytansinoids include C-19-dechloro (U.S. Pat. No. 4,256,746),C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,307,016 and 4,361,650), C-20-demethoxy (or C-20-acyloxy (—OCOR),+/−dechrolo (U.S. Pat. No. 4,294,757), C-9-SH (U.S. Pat. No. 4,424,219),C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598),C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254), C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866), C-15-methoxy(U.S. Pat. Nos. 4,313,946 and 4,315,929), C-18-N-demethyl (U.S. Pat.Nos. 4,362,663 and 4,322,348), and 4,5-deoxy (U.S. Pat. No. 4,371,533).

Various positions on maytansinoid compounds may be used as the linkageposition, depending upon the type of link desired. For example, forforming an ester linkage, the C-3 position having a hydroxyl group, theC-14 position modified with hydrozymethyl, the C-15 position modifiedwith a hydroxyl a group, and the C-20 position having a hydroxyl groupare all suitable (U.S. Pat. No. 5,208,020, RE39,151, and U.S. Pat. No.6,913,748; US Patent Appl. Pub. Nos. 20060167245 and 20070037972, and WO2009099728).

Preferred maytansinoids include those known in the art as DM1, DM3, andDM4 (US Pat. Appl. Pub. Nos. 2009030924 and 20050276812, incorporatedherein by reference).

ADCs containing maytansinoids, methods of making such ADCs, and theirtherapeutic use are disclosed in U.S. Pat. Nos. 5,208,020 and 5,416,064,US Pat. Appl. Pub. No. 20050276812, and WO 2009099728 (all incorporatedby reference herein). Linkers that are useful for making maytansinoidADCs are know in the art (U.S. Pat. No. 5,208,020 and US Pat. Appl. Pub.Nos. 2005016993 and 20090274713; all incorporated herein by reference).Maytansinoid ADCs comprising an SMCC linker may be prepared as disclosedin US Pat. Publ. No. 2005/0276812.

In certain embodiments, the ADC comprises an antibody conjugated to DM1with an SMCC linker. Preferred embodiments include Ab1-SMCC-DM1,Ab2-SMCC-DM1, Ab3-SMCC-DM1, Ab4-SMCC-DM1, Ab5-SMCC-DM1, Ab6-SMCC-DM1,Ab7-SMCC-DM1, and Ab8-SMCC-DM1.

Drug Loading

An ADC may have 1 to 20 chemotherapeutic agents per antibody.Compositions of ADCs may be characterized by the average number of drugmoieties per antibody molecule in the composition. The average number ofdrug moieties may be determined by conventional means such as massspectrometry, immunoassay, and HPLC. In some instances, a homogeneousADC population may be separated and purified by means of reverse phaseHPLC or electrophoresis. Thus, pharmaceutical ADC compositions maycontain a heterogeneous or homogeneous population of antibodies linkedto 1, 2, 3, 4, 5, 6, 7 or more drug moieties.

In preferred embodiments, the ADC comprises as antibody conjugated toone or more DM1 molecules. Embodiments of the invention includecompositions comprising an average of about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, or about 20 DM1 molecules per antibody. Preferred ADCcompositions are those comprising an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,or Ab8 having on average between 1 and 10 DM1 molecules per antibody,those comprising antibodies having on average between 3 and 7 DM1molecules per antibody, and those comprising antibodies having onaverage between 4 and 6 DM1 molecules, including an average of about4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6,about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9and about 6.0 DM1 molecules per antibody.

Effector Function-Enhanced Antibodies

One of the functions of the Fc portion of an antibody is to communicateto the immune system when the antibody binds its target. This isconsidered “effector function.” Communication leads toantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and/or complement dependent cytotoxicity(CDC). ADCC and ADCP are mediated through the binding of the Fc to Fcreceptors on the surface of cells of the immune system. CDC is mediatedthrough the binding of the Fc with proteins of the complement system,e.g., C1q.

The IgG subclasses vary in their ability to mediate effector functions.For example IgG1 is much superior to IgG2 and IgG4 at mediating ADCC andCDC. Thus, in embodiments wherein a cell expressing CD27L is targetedfor destruction, an anti-CD27L IgG1 antibody would be preferred.

The effector function of an antibody can be increased, or decreased, byintroducing one or more mutations into the Fc. Embodiments of theinvention include antigen binding proteins, e.g., antibodies, having anFc engineered to increase effector function (U.S. Pat. No. 7,317,091 andStrohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated hereinby reference in its entirety). Exemplary IgG1 Fc molecules havingincreased effector function include (based on the Kabat numberingscheme) those have the following substitutions: S239D/I332E

S239D/A330S/I332E

S239D/A330L/I332E

S298A/D333A/K334A

P247I/A339D

P247I/A339Q

D280H/K290S

D280H/K290S/S298D

D280H/K290S/S298V

F243L/R292P/Y300L

F243L/R292P/Y300L/P396L

F243L/R292P/Y300LN3051/P396L

G236A/S239D/I332E

K326A/E333A

K326W/E333S

K290E/S298G/T299A

K290N/S298G/T299A

K290E/S298G/T299A/K326E

K290N/S298G/T299A/K326E

Further embodiments of the invention include antigen binding proteins,e.g., antibodies, having an Fc engineered to decrease effector function.Exemplary Fc molecules having decreased effector function include (basedon the Kabat numbering scheme) those have the following substitutions:

N297A (IgG1)

L234A/L235A (IgG1)

V234A/G237A (IgG2)

L235A/G237A/E318A (IgG4)

H268QN309L/A330S/A331S (IgG2)

C220S/C226S/C229S/P238S (IgG1)

C226S/C229S/E233P/L234V/L235A (IgG1)

L234F/L235E/P331S (IgG1)

S267E/L328F (IgG1)

Another method of increasing effector function of IgG Fc-containingproteins is by reducing the fucosylation of the Fc. Removal of the corefucose from the biantennary complex-type oligosachharides attached tothe Fc greatly increased ADCC effector function without altering antigenbinding or CDC effector function. Several ways are known for reducing orabolishing fucosylation of Fc-containing molecules, e.g., antibodies.These include recombinant expression in certain mammalian cell linesincluding a FUT8 knockout cell line, variant CHO line Lec13, rathybridoma cell line YB2/0, a cell line comprising a small interferingRNA specifically against the FUT8 gene, and a cell line coexpressingB-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II.Alternatively, the Fc-containing molecule may be expressed in anon-mammalian cell such as a plant cell, yeast, or prokaryotic cell,e.g., E. coli. Thus, in certain embodiments of the invention, acomposition comprises an antibody, e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,Ab7, or Ab8, having reduced fucosylation or lacking fucosylationaltogether.

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of one or a plurality ofthe antigen binding proteins of the invention together with apharmaceutically effective diluents, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In certain embodiments, the antigenbinding protein is an antibody, including a drug-conjugated antibody ora bispecific antibody. Pharmaceutical compositions of the inventioninclude, but are not limited to, liquid, frozen, and lyophilizedcompositions.

Preferably, formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of a CD27L antigen binding protein, e.g, a CD27L-binding ADC, areprovided.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine, proline, or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins of the invention. In certain embodiments, theprimary vehicle or carrier in a pharmaceutical composition may be eitheraqueous or non-aqueous in nature. For example, a suitable vehicle orcarrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. In specific embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,and may further include sorbitol or a suitable substitute therefor. Incertain embodiments of the invention, CD27L antigen binding proteincompositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the CD27L antigen binding protein product may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art. The formulation components are present preferablyin concentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired CD27L antigen binding protein in a pharmaceutically acceptablevehicle. A particularly suitable vehicle for parenteral injection issterile distilled water in which the CD27L antigen binding protein isformulated as a sterile, isotonic solution, properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads or liposomes, that may provide controlled orsustained release of the product which can be delivered via depotinjection. In certain embodiments, hyaluronic acid may also be used,having the effect of promoting sustained duration in the circulation. Incertain embodiments, implantable drug delivery devices may be used tointroduce the desired antigen binding protein.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, CD27L antigen binding proteins areadvantageously formulated as a dry, inhalable powder. In specificembodiments, CD27L antigen binding protein inhalation solutions may alsobe formulated with a propellant for aerosol delivery. In certainembodiments, solutions may be nebulized. Pulmonary administration andformulation methods therefore are further described in InternationalPatent Application No. PCT/US94/001875, which is incorporated byreference and describes pulmonary delivery of chemically modifiedproteins.

It is also contemplated that formulations can be administered orally.CD27L antigen binding proteins that are administered in this fashion canbe formulated with or without carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofthe CD27L antigen binding protein. Diluents, flavorings, low meltingpoint waxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders may also be employed.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving CD27L antigen bindingproteins in sustained- or controlled-delivery formulations. Techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.See, for example, International Patent Application No. PCT/US93/00829,which is incorporated by reference and describes controlled release ofporous polymeric microparticles for delivery of pharmaceuticalcompositions. Sustained-release preparations may include semipermeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Sustained release matrices may include polyesters,hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 andEuropean Patent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Aspects of the invention includes self-buffering CD27L antigen bindingprotein formulations, which can be used as pharmaceutical compositions,as described in international patent application WO 06138181A2(PCT/US2006/022599), which is incorporated by reference in its entiretyherein.

As discussed above, certain embodiments provide CD27L antigen bindingproteins protein compositions, particularly pharmaceutical CD27L antigenbinding protein compositions, that comprise, in addition to the CD27Lantigen binding protein, one or more excipients such as thoseillustratively described in this section and elsewhere herein.Excipients can be used in the invention in this regard for a widevariety of purposes, such as adjusting physical, chemical, or biologicalproperties of formulations, such as adjustment of viscosity, and orprocesses of the invention to improve effectiveness and or to stabilizesuch formulations and processes against degradation and spoilage due to,for instance, stresses that occur during manufacturing, shipping,storage, pre-use preparation, administration, and thereafter.

A variety of expositions are available on protein stabilization andformulation materials and methods useful in this regard, such as Arakawaet al., “Solvent interactions in pharmaceutical formulations,” PharmRes. 8(3): 285-91 (1991); Kendrick et al., “Physical stabilization ofproteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEINFORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al.,“Surfactant-protein interactions,” Pharm Biotechnol. 13: 159-75 (2002),each of which is herein incorporated by reference in its entirety,particularly in parts pertinent to excipients and processes of the samefor self-buffering protein formulations in accordance with the currentinvention, especially as to protein pharmaceutical products andprocesses for veterinary and/or human medical uses.

Salts may be used in accordance with certain embodiments of theinvention to, for example, adjust the ionic strength and/or theisotonicity of a formulation and/or to improve the solubility and/orphysical stability of a protein or other ingredient of a composition inaccordance with the invention.

As is well known, ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface and by shieldingcharged and polar groups in the protein and reducing the strength oftheir electrostatic interactions, attractive, and repulsiveinteractions. Ions also can stabilize the denatured state of a proteinby binding to, in particular, the denatured peptide linkages (—CONH) ofthe protein. Furthermore, ionic interaction with charged and polargroups in a protein also can reduce intermolecular electrostaticinteractions and, thereby, prevent or reduce protein aggregation andinsolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating pharmaceuticalcompositions in accordance with the invention. One example is theHofmeister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic.” Destabilizingsolutes are referred to as “chaotropic.” Kosmotropes commonly are usedat high concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofmeister series.

Free amino acids can be used in CD27L antigen binding proteinformulations in accordance with various embodiments of the invention asbulking agents, stabilizers, and antioxidants, as well as other standarduses. Lysine, proline, serine, and alanine can be used for stabilizingproteins in a formulation. Glycine is useful in lyophilization to ensurecorrect cake structure and properties. Arginine may be useful to inhibitprotein aggregation, in both liquid and lyophilized formulations.Methionine is useful as an antioxidant.

Polyols include sugars, e.g., mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.

Among polyols useful in select embodiments of the invention is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations. It ensures structural stability to the cake. It isgenerally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucroseare among preferred agents for adjusting tonicity and as stabilizers toprotect against freeze-thaw stresses during transport or the preparationof bulks during the manufacturing process. Reducing sugars (whichcontain free aldehyde or ketone groups), such as glucose and lactose,can glycate surface lysine and arginine residues. Therefore, theygenerally are not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred polyols of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard.

Embodiments of the CD27L antigen binding protein formulations furthercomprise surfactants. Protein molecules may be susceptible to adsorptionon surfaces and to denaturation and consequent aggregation atair-liquid, solid-liquid, and liquid-liquid interfaces. These effectsgenerally scale inversely with protein concentration. These deleteriousinteractions generally scale inversely with protein concentration andtypically are exacerbated by physical agitation, such as that generatedduring the shipping and handling of a product.

Surfactants routinely are used to prevent, minimize, or reduce surfaceadsorption. Useful surfactants in the invention in this regard includepolysorbate 20, polysorbate 80, other fatty acid esters of sorbitanpolyethoxylates, and poloxamer 188.

Surfactants also are commonly used to control protein conformationalstability. The use of surfactants in this regard is protein-specificsince, any given surfactant typically will stabilize some proteins anddestabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine. Consequently,polysorbates should be used carefully, and when used, should be employedat their lowest effective concentration. In this regard, polysorbatesexemplify the general rule that excipients should be used in theirlowest effective concentrations.

Embodiments of CD27L antigen binding protein formulations furthercomprise one or more antioxidants. To some extent deleterious oxidationof proteins can be prevented in pharmaceutical formulations bymaintaining proper levels of ambient oxygen and temperature and byavoiding exposure to light. Antioxidant excipients can be used as wellto prevent oxidative degradation of proteins. Among useful antioxidantsin this regard are reducing agents, oxygen/free-radical scavengers, andchelating agents. Antioxidants for use in therapeutic proteinformulations in accordance with the invention preferably arewater-soluble and maintain their activity throughout the shelf life of aproduct. EDTA is a preferred antioxidant in accordance with theinvention in this regard.

Antioxidants can damage proteins. For instance, reducing agents, such asglutathione in particular, can disrupt intramolecular disulfidelinkages. Thus, antioxidants for use in the invention are selected to,among other things, eliminate or sufficiently reduce the possibility ofthemselves damaging proteins in the formulation.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease. Mg⁺², Mn⁺², and Zn⁺², however,can destabilize rhDNase. Similarly, Ca⁺² and Sr⁺² can stabilize FactorVIII, it can be destabilized by Mg⁺², Mn⁺² and Zn⁺², Cu⁺² and Fe⁺², andits aggregation can be increased by Al⁺³ ions.

Embodiments of the CD27L antigen binding protein formulations furthercomprise one or more preservatives. Preservatives are necessary whendeveloping multi-dose parenteral formulations that involve more than oneextraction from the same container. Their primary function is to inhibitmicrobial growth and ensure product sterility throughout the shelf-lifeor term of use of the drug product. Commonly used preservatives includebenzyl alcohol, phenol and m-cresol. Although preservatives have a longhistory of use with small-molecule parenterals, the development ofprotein formulations that includes preservatives can be challenging.Preservatives almost always have a destabilizing effect (aggregation) onproteins, and this has become a major factor in limiting their use inmulti-dose protein formulations. To date, most protein drugs have beenformulated for single-use only. However, when multi-dose formulationsare possible, they have the added advantage of enabling patientconvenience, and increased marketability. A good example is that ofhuman growth hormone (hGH) where the development of preservedformulations has led to commercialization of more convenient, multi-useinjection pen presentations. At least four such pen devices containingpreserved formulations of hGH are currently available on the market.Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech) &Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn)contain phenol while Somatrope (Eli Lilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation anddevelopment of preserved dosage forms. The effective preservativeconcentration in the drug product must be optimized. This requirestesting a given preservative in the dosage form with concentrationranges that confer anti-microbial effectiveness without compromisingprotein stability.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability should bemaintained over the entire product shelf-life (.about.18 to 24 months).An important point to note is that preservative effectiveness should bedemonstrated in the final formulation containing the active drug and allexcipient components.

CD27L antigen binding protein formulations generally will be designedfor specific routes and methods of administration, for specificadministration dosages and frequencies of administration, for specifictreatments of specific diseases, with ranges of bio-availability andpersistence, among other things. Formulations thus may be designed inaccordance with the invention for delivery by any suitable route,including but not limited to orally, aurally, opthalmically, rectally,and vaginally, and by parenteral routes, including intravenous andintraarterial injection, intramuscular injection, and subcutaneousinjection.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of a CD27L antigen bindingprotein-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication for which the CD27L antigen binding protein is beingused, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about30 mg/kg or more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 1.0 μg/kg up to about 20 mg/kg,optionally from 10 μg/kg up to about 10 mg/kg or from 100 μg/kg up toabout 5 mg/kg.

A therapeutic effective amount of a CD27L antigen binding proteinpreferably results in a decrease in severity of disease symptoms, inincrease in frequency or duration of disease symptom-free periods or aprevention of impairment or disability due to the disease affliction.For treating CD27L-expressing tumors, a therapeutically effective amountof CD27L antigen binding protein, e.g. an anti-CD27L ADC, preferablyinhibits cell growth or tumor growth by at least about 20%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, or at least about 90% relative to untreatedpatients. The ability of a compound to inhibit tumor growth may beevaluated in an animal model predictive of efficacy in human tumors.

Pharmaceutical compositions may be administered using a medical device.Examples of medical devices for administering pharmaceuticalcompositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196;4,447,224; 4,447, 233; 4,486,194; 4,487,603; 4,596,556; 4,790,824;4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163,all incorporated by reference herein.

Methods of Diagnosing or Treating a CD27L-associated Disease or Disorder

The CD27L antigen binding proteins of the invention are particularlyuseful for detecting CD27L in a biological sample. In certainembodiments, a biological sample obtained from a patient is contactedwith a CD27L antigen binding protein. Binding of the CD27L antigenbinding protein to CD27L is then detected to determine the presence orrelative amount of CD27L in the sample. Such methods may be useful indiagnosing or determining patients that are amenable to treatment with aCD27L antigen binding protein, e.g., an anti-CD27L ADC.

In certain embodiments, a CD27L antigen binding protein of the inventionis used to diagnose, detect, or treat an autoimmune or inflammatorydisorder. In treating autoimmune or inflammatory disorders, the CD27Lantigen binding protein may target CD27L-expressing cells of the immunesystem for destruction and/or may block the interaction of CD27L withthe receptor CD27.

CD27L interaction with CD27 is thought to play a role in cell-mediatedautoimmune diseases, such as experimental autoimmune encephalomyelitis(EAE) (Nakajima et al. (2000) J. Neuroimmunol. 109:188-96). This effectis thought to be mediated in part by an inhibition of TNF-a production.Furthermore, blocking of CD27L signaling inhibits CD40-mediated clonalexpansion of CD8+ T-cells and reduces the generation of CD8+ memoryT-cells (Taraban et al. (2004) J. Immunol. 173:6542-6). As such, theCD27L antigen binding proteins can be used to treat a subject with anautoimmune disorder, e.g., a disorder characterized by the presence ofB-cells expressing CD27L including, for example, experimental autoimmuneencephalomyelitis. Additional autoimmune disorders in which theantibodies of this disclosure can be used include, but are not limitedto systemic lupus erythematosus (SLE), insulin dependent diabetesmellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn'sDisease, ulcerative colitis and Celiac disease), multiple sclerosis(MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) andglomerulonephritis. Furthermore, the cd27L antigen binding proteincompositions of this disclosure can be used for inhibiting or preventingtransplant rejection or in the treatment of graft versus host disease(GVHD).

Additionally, the interaction of CD27L with CD27 has also been proposedto play a role in signaling on CD4+ T cells. Some viruses have beenshown to signal the CD27 pathway, leading to destruction of neutralizingantibody responses (Matter et al. (2006) J Exp Med 203:2145-55). Assuch, the CD27L antigen binding protein compositions and methods of thepresent disclosure can be used to treat a subject with a viral infectionincluding, for example, infections from human immunodeficiency virus(HIV), Hepatitis (A, B, & C), Herpesvirus, (e.g., VZV, HSV-1, HAV-6,HSV-II and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus andarboviral encephalitis virus and lymphocytic choriomeningitis virus(LCMV) or in the treatment of HIV infection/AIDS. Additionally, thehuman antibodies, antibody compositions and methods of the presentdisclosure can be used to inhibit TNF-a production.

In certain embodiments, a CD27L antigen binding protein of the inventionis used to diagnose, detect, or treat a cancer or tumorigenic disorder.Tumors and cancers amenable for treatment herein, wherein the cells ofthe tumor or cancer may express CD27L include, but are not limited to:renal cell carcinomas (RCC), such as clear cell RCC, papillary RCC,chromophobe RCC, and the like, glioblastoma, Head and Neck Cancers(e.g., squamous cell carcinomas (HNSCC), and the like), breast cancer,brain tumors, nasopharangeal carcinomas, non-Hodgkin's lymphoma (NHL),such as low grade NHL, diffuse large cell NHL, and the like, acutelymphocytic leukemia (ALL), such as pre-B-ALL, and the like, chroniclymphocytic leukemia (CLL or B-CLL), Burkitt's lymphoma, anaplasticlarge-cell lymphomas (ALCL), multiple myeloma, cutaneous T-celllymphomas, nodular small cleaved-cell lymphomas, lymphocytic lymphomas,peripheral T-cell lymphomas, Lennert's lymphomas, immunoblasticlymphomas, T-cell leukemia/lymphomas (ATLL), adult T-cell leukemia(T-ALL), entroblastic/centrocytic (cb/cc) follicular lymphomas cancers,diffuse large cell lymphomas of B lineage, angioimmunoblasticlymphadenopathy (AILD)-like T cell lymphoma, HIV associated body cavitybased lymphomas, embryonal carcinomas, undifferentiated carcinomas ofthe rhino-pharynx (e.g., Schmincke's tumor), Castleman's disease,Kaposi's Sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia andother B-cell lymphomas. Additional tumor types amenable for treatmentherein, include tumors of the: kidney, pancreas, larynx or pharynx,melanoma, ovary, lung adenocarcinoma, colon breast, brain, and the like.

EXAMPLES

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the presentinvention and are intended to limit its scope.

Example 1—Fully Human Monoclonal Antibodies Against CD27L

The generation of fully human antibodies directed against human CD27Lwas carried out using XENOMOUSE® technology (U.S. Pat. Nos. 6,114,598;6,162,963; 6,833,268; 7,049,426; 7,064,244, which are incorporatedherein by reference in their entirety; Green et al., 1994, NatureGenetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Greenand Jakobovitis, 1998, J. Ex. Med. 188:483-495).

IgG1, IgG2, and IgG4 XENOMOUSE® mice were immunized/boosted with solublehuman CD27L or human CD27L recombinantly expressed on the surface ofChinese Hamster Ovary (CHO) cells. Hybridomas were produced from theimmunized mice. Hybridoma supernatants were screened for binding torecombinant human CD27L-expressing 293 cells. Over 260 supernatants werepositive for binding.

The positive supernatants were then screened for binding to native CD27Lon the surface of Raji and/or 786-0 cells. The native CD27L bindingassay revealed 161 positive supernatants.

Those supernatants were tested for cross-reactivity with cynomolgusCD27L. Twenty-five supernatants were positive for cross-reactivity withcynomolgus monkey CD27L. Those twenty five were then tested for CDCactivity and the ability to block human CD27 binding to human CD27L.Nineteen supernatants were positive for CDC activity and for inhibitionof CD27 binding to CD27L. Subcloning and sequencing of the 13 IgG1 and 6IgG4 lines revealed 7 unique IgG1 antibodies (Ab1-Ab7) and 1 unique IgG4antibody (Ab8).

A summary of the characteristics of the eight antibodies along withthose of a chimeric version of a commercially available mouse anti-humanCD27L antibody are provided in FIG. 1.

Affinity

Affinity for recombinant soluble human CD27L was determined using a CMSchip on a BIACORE 3000. Goat anti-human IgG antibody was used to capturethe test antibody. Binding and dissociation of Histidine-tagged humansoluble CD27L was measured to determine K_(a), K_(d), and K_(D). Theconditions were as follows:

Temperature=25° C.

Flow Rate=50 ul/min

Running Buffer=HBS-EP

Regenerations with 10 mM Glycine pH 1.5

Concentration range of Hu CD27L-his were 200 nM→0.217 nM

Model Fit (Scrubber2): 1:1 Binding+local Rmax

5 Minute Association and 25 Minute Dissociation

ADCC Activity

The ADCC assay was performed in a sterile 96 well round bottom tissueculture plate (Corning). Antibodies were titrated from 20 μg/mL to0.0002 μg/mL by carrying 10 μL in 1004 of complete RPMI containing 10%FCS (a 1:10 dilution). Calcein-labeled targets were added, 50 μL tocontain 10,000 cells. Target cells and various concentrations ofantibody were incubated for 40 minutes at 4° C., then NK cell effectorsadded, 50 μL to contain 200,000 cells. Cultures were incubated for 4 hrsat 37° C. then supernatants pulled and assayed for calcein release bymeasuring fluorescence at 485-535 nm on a Wallac Victor II 1420Multilable HTS counter. 100% lysis values were determined by lysing sixwells of calcein labeled Raji targets with Igepal 630 detergent (3 μLper well) and spontaneous lysis values determined by measuring thefluorescence in supernatants from targets alone.

Percent (%) specific lysis was defined as (samplefluorescence)−(spontaneous lysis fluorescence)/(100% lysis−spontaneouslysis fluorescence). Raw data was entered in an Excel spreadsheet withthe embedded formulae to calculate % specific lysis and resultant valuestransferred to graphic program (GraphPad Prism) where the data wastransformed in a sigmoidal curve fit graph. Subsequent analyses (linearregression calculations) were done in GraphPad to generate EC₅₀ values.

ADCP Activity

Monocytes were negative selected from human peripheral blood and storedin 4° C. cold room over night with medium RPMI 1640 containing 10% FBS.Then monocytes were seeded to a 48-well tissue culture plate at 200,000cells per well with 200 μL of growth medium (RPMI 1640 containing 10%FBS and 40 ng/ml Hu M-CSF) and incubated at 37° C., 5% CO₂ for 6 days tolet monocytes differentiate to macrophages.

On Day 6, the ADCP assay was performed as follows:

-   -   1. Labeling target cells with PKH67 green dye at final        concentrations of 2×10⁻⁶ M PKH67 dye        -   Tumor cells were collected and washed once with PBS by            centrifuging the cells (400′ g) for 5 minutes.        -   After centrifuging cells, the supernatant was carefully            aspirated, but leaving no more than 25 mL of supernatant.        -   Four μL of the PKH67 ethanolic dye solution at stock            concentration of 4×10⁻⁶ M was added to 1 ml of Diluent C            from kit in polypropylene tube and mixed well.        -   Cell pellets were re-suspended into 1 mL of Diluent C at a            density of 20×10⁶ in polypropylene tube.        -   Cells were rapidly transferred to dye work solution with            gently pipetting to insure complete dispersion.        -   The mixture was incubated at room temperature for 4 minutes            with mixing periodically.        -   Two mL of whole activated FBS was added into cells to stop            the staining and incubated at room temp for 1 minute to            allow binding of excess dye.        -   Forty mL of RPMI containing 10% FBS was added into cells and            washed once by centrifuging the cells (400′ g) for 10            minutes.        -   Cell pellets were suspended with 40 mL of medium again and            transferred to a new tube.        -   Cells were washed again three times with medium RPMI+10% FBS            and 1× with complete growth medium (RPMI 1640 containing 10%            FBS and 40 ng/ml Hu M-CSF).        -   Cells were counted and suspended with growth medium at 1×10⁶            cells per mL for T:E at 1:2 ratio.    -   2. Treatment of tumor cells with antibodies for antibody        dependent cellular phagocytosis (ADCP)        -   Antibody dilutions were prepared in macrophage growth            medium. These dilutions were concentrated at four times            higher than final concentrations.        -   To preincubate PKH67 green labeled target cells with            antibodies, 280 ul of 4× concentrated antibodies was mixed            with 280 ul of green labeled tumor cells and incubated at            4° C. for 30 minutes.        -   The mixture of green labeled tumor cells with anti-tumor            antibodies was added to macrophage cells in 48-well plate at            200 μl for each well as indicated in the Experiment Design            table below. The final volume is 0.4 ml per well. The ratio            of target cells to effect cells (macrophages) is 1:2.        -   Cells were incubated at 37° C., 5% CO₂ for one hour.    -   3. Counterstaining macrophages with macrophages marker        -   Target cells and macrophages in 48-well plate were detached            with Trypsin-Versene mixture.        -   Cells were transferred into a 96-well block with 2.2-ml            volume per well and washed once with pre-warmed FASC wash            solution by spinning the blocks at 400′ g for 5 minutes and            then discarding supernatant.        -   Macrophages were stained with their marker, CD11b-Biotin at            1:200 dilution in block solution with 100 μl per well for 10            min on ice.        -   After washing cells once, macrophages were detected with            streptavidin Alexa 568 at 1:1000 dilutions for 10 minute on            ice.        -   After washing cells 1× with PBS, cells were fixed with 4%            formaldehyde-PBS at room temperature for 20 minutes. Then            cells were washed 1× with dH2O.        -   Cell pellets were resuspended with water at 200 μl per well            and transferred to a 96-well plate at 100 μl per well.    -   4. Quantitative measurement of phagocytosis activity on an        ArrayScan V^(TI) HCS reader (version 6, Cellomics Inc. Thermo        Fisher Scientific, Pittsburgh, Pa.) with Target Activation        BioApplication employing a 20× objective. The filter setting was        indicated in Table 2. At least 200 cells were counted in each        well.

TABLE 2 Channel Target Label Fluor 1 macrophages Ms-anti-Hu CD11b redBiotin→streptavidin Alexa 568 2 Tumor cells PHK67 green

Statistical analysis was performed using Prism 4.01 (GraphPad, SanDiego, Calif.). A plot shows % tumor cell phagocytosis vs. the log ofantibody concentration in ng/ml. The percentage of tumor cellphagocytosis is represented with percentage of tumor cells that wereoverlapped with macrophages vs. total macrophages in the selected fieldsand obtained from output feature of ArrayScan reader “% ObjectCounts”.The % values were expressed as the mean+/−standard error of the mean(SEM) for duplicate measurements (n=2). The EC₅₀ was determined by usingnonlinear regression (curve fit) followed by using Sigmoidaldose-response equation. Data were normalized to the maximum and minimumsignal and fit to a sigmoidal dose-response curve.

CDC Activity

Preparation of Tumor Cells:

Raji cells were washed once with assay medium and resuspended in assaymedium (RPMI 1640 plus 1% FBS). Cells were seeded in a 96-well tissueculture plate at 50 μL per well with two cell densities for both rabbitand human complement complements. For rabbit complement, the celldensity was 5×10⁴ cells per well. For human complement, the cell densitywas 2×10⁵ cells per well.

Treatment of Cells with Complement and Test Antibody:

Three times concentrated complements were prepared in assay medium asoutlined in Table 3. Then they were added to cells in plates at 50 μLper well. For cells treated with rabbit complement, the finalconcentration of rabbit complement was 10%; for cells treated with humancomplement, the final concentration of human complement was 20%.

TABLE 3 Preparation of 3x concentrated complement in assay medium (RPMI1640 + 1% FBS) Complement Assay Total 3x Complement (ml) Medium (ml)Volume (ml) 30% HI rabbit C′ (inactive) 0.5 1.16 1.66 30% no HI rabbitC′ (active) 2.5 5.83 8.33 60% HI Hu C′ (inactive) 0.5 0.33 0.83 60% noHI Hu C′ (active) 3 2.0 5.0

Test antibodies at 10 μg/mL were added to cells at 50 μL per well. Thetotal volume in each well at the start of culture was 150 μL. Cells werecontinuously incubated at 37° C., 5% CO₂ for one hour for cells treatedwith rabbit complement and 6 hours for cells treated with humancomplement.

Measurement of Cytotoxicity with ArrayScan Plate Reader:

After incubation, 50 μl medium from each well was removed. The cocktailof Hoechst 33342 and propidium iodide (PI) which was prepared at 1:1000dilution in PBS solution containing 2% FBS was added into cells at 100μL per well. Cells treated with human complement were transferred into anew 96-well plate at 40 μL per well after gently mixing. Samples wereanalyzed on an ArrayScan V^(TI) HCS reader (version 6, Cellomics, ThermoFisher Scientific, Pittsburgh, Pa.) with BioApplication “TargetActivation” employing a 20× objective. The filter setting was indicatedin Table 4. At least 200 cells were counted in each well.

TABLE 4 Channel Target Label Fluor Filter 1 Nucleic acid for Hoechst33342 UV/460 nm DAPI all cells 2 Nucleic acid for Propidium iodide488/>575 nm TRITC dead cells

Statistical Analysis:

Statistical analysis was performed using Prism 4.01 (GraphPad, SanDiego, Calif.).

Internalization Time

Seeding cells: 786-0 cells were seeded on a 96-well plate at 10,000cells per well with 100 μL of growth medium (RPMI containing 10% FBS)and incubated at 37° C., 5% CO₂ for 2 days to reach to 100% cellconfluency on assay day. Plated cells were evaluated for 1)internalization and 2) endosomal co-localization.

Staining cells for internalization time-course: Plate was washed 1× withassay medium (PBS containing 2% FBS). Antibodies were added to wells at2 μg/mL per well (100 μL per well) in assay medium. Human antibodieswere allowed to bind to cells at 4° C. for 30 minutes and then washed 1×with assay medium. Anti-human IgG Fab′ Alexa 488 (1:100) and Hoechst33342 (1:2000) were added to cells in assay medium at 4° C. for 20minutes. Next cells were washed 1× with assay medium. Cells were eitherfixed and permeabilized, starting with time zero or incubated at 37° C.,5% CO₂ for 1, 3, or 5 hours. Post incubation, cells were fixed andpermeabilized with the following procedure. Cells were washed 1× with BDwash buffer, followed by addition of Fix/perm solution to wells at 100μL per well. Samples were analyzed on an ArrayScan V^(TI) HCS reader(version 6, Cellomics, Thermo Fisher Scientific, Pittsburgh, Pa.) withBioApplication “Spot Detector” employing a 40× objective. The filtersetting for internalization was indicated in Table 5. At least 400 cellswere counted in each well.

TABLE 5 Channel Target Label Filter 1 Nucleic acid Hoechst 33342 DAPI(blue) for all cells 2 Internalized spots Alexa 488 FITC (green)

Count-staining cells for co-localizing internalized spots to the earlyendosomal compartment: After internalization analysis, 786-0 cells werewashed 1× with BD buffer. Cells were explored to Fix/perm solution at RTfor 20 minutes. Following 2× wash steps, cells were incubated with EEA-1in BD buffer at 0.5 μg/mL (100 μl/well) at RT for 20 minutes. Cells werewashed 1× with BD buffer and added with anti-mouse Alexa 568 at 1:1000dilution. Cells were incubated at RT for 20 minutes followed by two washsteps of BD buffer solution.

Photographing images: Cell images for internalization andco-localization were taken with a Leica florescent microscope connectedto Hamamutsu digital camera with Openlab Image Analysis software(Improvision Inc, Lexington, Mass.) or ArrayScan V^(TI) HCS reader.

Statistical Analysis: Statistical analysis was performed using Prism4.01 (GraphPad, San Diego, Calif.). The spot counts were expressed asthe mean±standard error of the mean (SEM) for duplicate measurements(n=2). Using analysis tool, spot count per unit time (internalizationrate) was fit to a one phase exponential association equation.

Example 2—Assessment of MCC-DM1-Conjugated Antibodies

Ab1, Ab2, Ab4, Ab7, and Ab8 were conjugated to MCC-DM1 (see FIG. 12).The targeted load level was 4.5-5 drugs per antibody. Briefly, thelysines of the antibody were conjugated to the NHS-ester of thehetero-bifunctional cross-linker Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) which contains an NHS-ester and amaleimide. Next the linker-modified antibody was purified from excesslinker and then conjugated to the warhead DM1 via a sulfhydryl presenton DM1. Excess DM1 was then removed by a second purification step togenerate the final conjugate Ab-MCC-DM1.

More particularly, CD27L antibody, transiently expressed in mammaliancell culture 2936-E cells, was loaded onto a MabSelect SuRe column (GEHealthcare) that had been equilibrated in 25 mM Tris, 150 mM SodiumChloride, pH 7.4. The column with bound CD27L antibody was then washedwith 3 wash steps: first an equilibration buffer wash, followed by a 25mM Tris, 500 mM L-Arginine, pH 7.5 wash and a final wash withequilibration buffer. CD27L antibody was eluted with 100 mM SodiumAcetate, pH 3.5. Fractions containing the antibody were pooled andadjusted to a final pH of 5.0 with 1M Tris, pH 8.0. The antibody wassubsequently purified on a Fractogel® EMD SO3-(M) column (EMD ChemicalsInc) equilibrated in 30 mM Sodium Acetate, pH 5.0. Bound antibody waseluted with an 8CV gradient between 0 to 0.8M Sodium Chloride in 30 mMSodium Acetate, pH 5.0. Antibody containing fractions were pooled anddialyzed into Conjugation Buffer (2 mM EDTA, 50 mM Sodium Chloride, 50mM Potassium Phosphate, pH 6.5).

The purified CD27L antibody was modified with the amine reactive linkerSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)(Thermo Scientific) to introduce thiol reactive maleimide groups. Theantibody at a concentration of 55 μM was treated with 20 molarequivalents of SMCC in Conjugation Buffer adjusted to 10%dimethylacetamide (v/v) in the final reaction mixture. After incubationfor 90 minutes at room temperature, the reaction mixture was desaltedwith a HiPrep 26/10 Desalting Column containing Sephadex G-25 fine resin(GE Healthcare) equilibrated in 2 mM EDTA, 150 mM Sodium Chloride, 35 mMSodium Citrate, pH 5.0. Antibody containing fractions were pooled andassayed for degree of modification with linker using Ellman's reagent(5,5′-dithio-bis-[2-nitrobenzoic acid]) as described below. The antibodywas found to be modified with an average of 7.5 maleimide groups perantibody.

Ellman's reagent is cleaved by thiols, yielding a yellow product with anabsorbance at 412 nm. A subtractive Ellman's assay was used to determinethe number of maleimide groups on the CD27L antibody after reaction withSMCC. CD27L antibody modified with SMCC or a control sample of bufferwithout antibody was incubated with an equivalent concentration ofthiols, 0.4 mM DTT (dithiothreitol). Any maleimides present in theantibody sample would react with the thiols in DTT, making itunavailable for further reaction with Ellman's reagent. Ellman's reagentwas then added to both samples and a colorimetric quantitation at 412 nmwas made to determine the concentration of reacted Ellman's reagent. Thedecreased concentration of thiols in the antibody sample as compared tothe control sample is proportional to the number of maleimides presentin the antibody sample. This value was used to determine the number oflinked maleimide groups per modified CD27L antibody.

The SMCC modified CD27L antibody (7.5 maleimide groups per antibody) ata concentration between 17 μM-27 μM was treated with 1.7 molarequivalents of DM1 (Immunogen) per maleimide group buffered with 2 mMEDTA, 150 mM NaCl, 35 mM Sodium Citrate, pH 5.0 adjusted to 3% DMA (v/v)in the final reaction mixture. Reaction mixtures were incubated at roomtemperature overnight for up to 20 hours. The reaction mixture wasloaded on a Superdex 200 gel filtration column (GE Healthcare)equilibrated with 20 mM Sodium Phosphate, 150 mM Sodium Chloride, pH6.5. Fractions were collected and monomeric antibody containingfractions pooled and assayed. The molar ratio of DM1 molecules linkedper antibody was determined by measuring the absorbance at 252 nm and280 nm, and found to be 4.5-5.0 DM1 molecules per antibody.

In Vitro Pharmacology

The antibody drug conjugates (ADC) Ab4- and Ab8-MCC-DM1 demonstratedcomparable native CD27L binding to their un-conjugated counterparts asassessed by flow cytometry. The observed binding EC50 for Ab4 andAb4-MCC-DM1 were the same while it was a modest 4-fold lower forAb8-MCC-DM1 as compared to Ab8 (Table 6). A more precise measure ofbinding affinity was determined for Ab4, Ab8 and their conjugatecounterparts by measuring their ability to bind native human CD27Lexpressed on Raji cells using Gyros technology. The results showed thatboth conjugates exhibited sub-nM affinities for CD27L within two-fold ofthose observed for the un-conjugated Ab4 and Ab8 (Table 6). Bothunconjugated and conjugated Ab4 and Ab8 also exhibited bindingaffinities to soluble human CD27L-his within 2-fold of each other asdetermined by BIACORE.

TABLE 6 Binding comparison of Ab4 and Ab8 to their MCC-DM1 conjugatecounterparts Ab4- Ab8- Measurement Ab4 MCC-DM1 Ab8 MCC-DM1 Gyros 0.014nM 0.023 nM 0.078 nM 0.077 nM K_(D)-native CD27L-Raji FACS  0.1 nM  0.1nM  0.02 nM  0.08 nM (EC₅₀)-786-0

The internalization of Ab4-MCC-DM1, Ab8-MCC-DM1, Ab4, and Ab8 wasevaluated in the CD27L-expressing human ccRCC line 786-0. CD27Lexpressing 786-0 cells were exposed to un-conjugated Ab4 and Ab8 humananti-CD27L antibody, Ab4-MCC-DM1, Ab8-MCC-DM1, control HuIgG1, orcontrol αSA-MCC-DM1 and allowed to bind at 4° C. Internalization of thetest articles was evaluated using FluoroNanogold goat anti-hu IgG Fab′Alexa 488. Cells were fixed and permeabilized at specific times afterincubation with test articles (time=0, 1, 3, and 5 hour) and imagedusing an ArrayScan VTI HCS reader. Co-localization of internalized testarticles to endosomes was determined using anti-EEA-1 antibody, anendosomal marker. The time-dependent internalization of the antibodies,including Ab4-MCC-DM1, was observed via fluorescence microscopy imageanalysis by the formation of punctate spots within the cellularcytoplasm at 37° C. compared with cell membrane localization at timezero at 4° C. Ab4-MCC-DM1 co-localized with the endosomal marker EEA-1after a 5-hour incubation at 37° C., demonstrating that Ab4-MCC-DM1 isinternalized into the endosomal subcellular compartment of 786-0 cells.The level and rate of internalization was within a similar range forAb4, Ab8 and their conjugated counter-parts (FIG. 2).

Both Ab4-MCC-DM1 and Ab8-MCC-DM1 demonstrated potent and specific invitro growth inhibition of CD27L-expressing tumor cells. In ananti-proliferation (tumor growth inhibition) assay, Ab4-MCC-DM1,Ab8-MCC-DM1 or un-conjugated anti-CD27L Ab4 or Ab8 were incubated withCD27L expressing 786-0 luciferase or CD27L negative H1650 target cellsin the presence/absence of naked Ab4 or Ab8, respectively, or controlHuIgG1 for 4 days. Both cell lines, 786-0 luciferase and H1650, wereseeded in 96-well tissue culture plates with growth medium at 100 μL ofwell with 500 cells per well for 786-0 luciferase and 1000 cells perwell for H1650. All plates were incubated at 37° C., 5% CO2 for 4 hours.After a 4 hour incubation, naked Ab and conjugates were added to cellsat 100 μL per well at various dose titrations. The total volume in eachwell at the start of culture was 200 μL. Cells were continuouslyincubated at 37° C., 5% CO2 for 4 days prior to measurement of cellularATP levels. To assess cell growth inhibition, ATP levels (as a measureof cell number) were measured via luminescence using the CellTiter-Gloassay kit.

Both Ab4-MCC-DM1 and Ab8-MCC-DM1 inhibited cell growth ofCD27L-expressing 786-0 cells with a concentration of 50% inhibition(IC50) as set forth in Table 7. Cell growth inhibition was not observedin CD27L-negative H1650 cells upon exposure to Ab4-MCC-DM1 andAb8-MCC-DM1. Addition of an excess of unconjugated parental anti-CD27Lantibody was able to block Ab4-MCC-DM1 mediated growth inhibition,confirming CD27L binding by Ab4-MCC-DM1 was required for activity.Unconjugated parental anti-CD27L antibody did not inhibit the growth ofCD27L-expressing 786-0 cells. Control conjugate,anti-streptavidin-MCC-DM1 (αSA-MCC-DM1), did not exhibit any inhibitionof cell growth in either the CD27L-positive or the CD27L-negative cellline. The results indicate that both Ab4-MCC-DM1 and Ab8-MCC-DM1 werepotent inhibitors of 786-0 cell growth compared to control conjugate(FIG. 3). Ab4-MCC-DM1 tended to show a slight increase in potency overAb8-MCC-DM1 (Table 7).

TABLE 7 Cellular Potency of anti CD27L-MCC-DM1 conjugates 786-0-_(IC50)Ab4-MCC-DM1 Ab8-MCC-DM1 Drug Conc. (nM) 0.34 0.54 Antibody Conc. (nM)0.07 0.11

Ab4-MCC-DM1 mediated Antibody Dependent Cell Cytotoxicity (ADCC) againstRaji cells with similar potency (EC50=0.006 μg/mL) and magnitude to thatobserved for the unconjugated parental Ab4 anti-CD27L antibody(EC50=0.01 μg/mL). Briefly, Natural Killer (NK) cells isolated from thePBMC obtained from a normal human blood donor were incubated withcalcein labeled Raji human B cell lymphoma target cells (express CD27L)in the presence of Ab4-MCC-DM1 or control antibodies as described above.Percent (%) specific cytotoxicity was determined by measuring calceinrelease from CD27L expressing target cells lysed in the presence ofAb4-MCC-DM1 as compared to control wells. The results are shown in FIG.8 which indicate that Ab4-MCC-DM1 mediated a similar level of ADCC tothat of the Ab4 antibody (EC 50 values of 0.006 and 0.01 μg/mLrespectively). The huIgG1 control did not mediate any measurable lysisof CD27L expressing targets. Both the Ab4-MCC-DM1 and the unconjugatedAb4 antibody are capable of inducing NK cell mediated ADCC of CD27Lspecific targets at similar levels. Similar results were observed forAb8-MCC-DM1 and the unconjugated Ab8 antibody.

vitro Antibody Dependent Cellular Phagocytosis (ADCP) activity ofAb4-MCC-DM1 or unconjugated parental antibody Ab4 was measured againstboth Raji and 786-0 tumor cells. Ab4-MCC-DM1 mediated a similar level ofcomplement-mediated lysis to that observed for unconjugated parentalantibody Ab4. Briefly, macrophages were differentiated from monocytesisolated from the human peripheral blood obtained from normal humanblood donors. Macrophages were incubated with PHK67 green labeled CD27Lexpressing cell lines, 786-0 and Raji as target cells in the presence ofunconjugated anti CD27L antibody Ab4, HuIgG1, Ab4-MCC-DM1, or controlantibodies (αSA-MCC-DM1), as described above. Percent (%) tumor cellphagocytosis was determined from the percentage of tumor cells that wereengulfed by macrophages vs. total macrophages in the selected fields.The results are shown in FIG. 9 and indicate that Ab4-MCC-DM1 mediatedsimilar ADCP potency in CD27L expressing cell lines, 786-0 and Raji.EC50 values of the unconjugated Ab4 were within 10-fold of thoseobserved for Ab4-MCC-DM1 (see Table 8).

TABLE 8 786-0 Raji Abs EC50 (pM) EC50 (pM) Ab4 0.008 0.008 Ab4-MCC-DM10.087 1.421

Given that the dilution curve was performed at 1:40 dilution intervals,the observed EC50 for both Ab4-MCC-DM1 and the unconjugated Ab4 are of asimilar potency (with-in the margin of the dilution factor). Thus, boththe conjugated Ab4-MCC-DM1 and the unconjugated WT Ab4 antibodies arecapable of inducing human macrophage to mediate ADCP of CD27L expressingcells at similar levels. Similar results were observed for Ab8-MCC-DM1and the unconjugated Ab8 antibody.

In vitro Complement Dependent Cytotoxicity (CDC) activity of Ab4-MCC-DM1was assessed employing both human and rabbit complement andCD27L-expressing Raji tumor cells. At 10 μg/mL, Ab4-MCC-DM1 mediated asimilar level of complement-mediated lysis to that observed forunconjugated parental antibody Ab4 (rabbit complement 72% lysis andhuman 17% lysis). Briefly, activated rabbit or human complements wereincubated with CD27L expressing Raji target cells in the presence ofanti CD27L antibodies or control antibodies, as described above. CDCmediated killing was measured from output feature of ArrayScan reader “%selected objects” to detect % of PI positive vs. Hoechst for “%Cytotoxicity.” The results indicate that Ab4-MCC-DM1 mediated similarlevels of CDC to that of the WT Ab4 antibody when incubating tumor Rajicells with 10% baby rabbit complement or 20% human complement. Heatinactivated rabbit or human complements didn't show any CDC mediatedkilling against Raji. Thus, both Ab4-MCC-DM1 and un-conjugated Ab4induce a similar level of CDC activity against CD27L expressing tumortarget cells. Similar results were observed for Ab8-MCC-DM1 and theunconjugated Ab8 antibody.

In Vivo Pharmacology

In vivo passaged 786-0 ccRCC cells (786-0 S4) were implanted into femaleCB-17/SCID mice using growth-factor reduced MATRIGEL to generate tumorxenografts for efficacy studies. 786-0 S4 cells express an average ofapproximately 180,000 CD27L sites/cell. Treatment with Ab4-MCC-DM1 orAb8-MCC-DM1 was initiated when the tumor size reached an average ofapproximately 250 mm³. Tumor-bearing animals were randomized by tumorsize into groups of ten animals each and dosed intravenously once. Ablinded dose response study of Ab4-MCC-DM1 and Ab8-MCC-DM1 employingdoses ranging from 7-64 ug DM1/kg (0.3-2.5 mg Ab/kg) was performed inthis established tumor model. Robust tumor regression was observed atthe low 7 ug DM1/kg, mid 25 ug DM1/kg and the high 64 ug DM1/kg doses.At the mid and high dose, complete regressions were maintained at least28 days following a single dose (FIG. 4). No body weight loss wasobserved in any of the dosing groups over the course of the study.

In another demonstration of in vivo efficacy, Caki-1 ccRCC cells thatexpress an average of 59,000 CD27L sites per cell (3-fold less that786-0 cells) were implanted into CB-17/SCID mice as described above.When the Caki-1 xenografts reached an average size of 250 mm³,tumor-bearing animals were randomized by tumor size into groups of tenanimals each and dosed intravenously once per week for 3 weeks (to moreclosely mimic a weekly clinical dosing regimen). A blinded dose responsestudy of Ab4-MCC-DM1 and Ab8-MCC-DM1 employing doses ranging from 60-270ug DM1/kg (2.4-11 mg Ab/kg) was performed in this established tumormodel. Tumor regression was initially observed at the mid 120 ug DM1/kgand the high 270 ug DM1/kg doses while tumor growth inhibition wasobserved at the low 60 ug DM1/kg dose compared to the control conjugateor vehicle. As time progressed, tumor regression in the two higher dosegroups began to diminish within 7 days of receiving the last dose (FIG.5).

Like ccRCC cells, Raji B-lymphoma cells express CD27L (about 200,000sites/cell) and internalize the anti-CD27L drug conjugate which resultsin mitotic arrest and cell death in vitro. Ab4-MCC-DM1 was evaluated forefficacy in the subcutaneous Raji Xenograft model. When the Rajixenografts reached an average size of 250 mm³, tumor-bearing animalswere randomized by tumor size into groups of ten animals each and dosedintravenously q4 days×2 and then once per week for one additional weekfor a total of 3 doses (to more closely mimic a weekly clinical dosingregimen). A blinded dose response study of Ab4-MCC-DM1 using dosesranging from 60-270 ug DM1/kg (2.4-11 mg Ab/kg) was performed in thisestablished tumor model. Tumor growth inhibition was observed at alldose levels, with >90% tumor growth inhibition being observed at the mid120 ug DM1/kg and the high 270 ug DM1/Kg doses during the active dosingperiod with an intermediate anti tumor response being observed at thelow 60 ug DM1/kg dose compared to control conjugate (FIG. 6). At the endof the tumor measurement period a slight majority of animals in the midand high dose groups exhibited tumor regression suggesting that furtherdose and schedule optimization could improve response.

The efficacy of different dosing intervals of control conjugateαSA-MCC-DM1, or Ab4-MCC-DM1 was evaluated in the 786-0 xenograft model.CB-17/SCID mice were implanted with 786-0 tumor cells. On day 13, 50animals were randomized into cohorts of 10 animals each with an averagetumor volume of 200 mm3. Each cohort was administered an intraperitonealdose of either control conjugate αSA-MCC-DM1 or Ab4-MCC-DM1. Tumorvolume is represented as group average±standard error of the mean (SEM).Statistical significance of observed differences between growth curveswas evaluated from days 13 to 59 by repeated measures analysis ofcovariance (RMANOVA) of the log transformed tumor volume data withDunnett adjusted multiple comparisons. Significance is attained ifp<0.05. Three doselevels of Ab4-MCC-DM1 (1.0, 2.0, or 2.9 mg/kg ofAb4-MCC-DM1 based upon antibody or 25, 50, or 75 μg/kg based upon DM1equivalents, respectively) were administered. The 1.0 mg/kg dose wasadministered once per week or once every 2 weeks for 6 weeks, the 2.0mg/kg dose was administered once every 2 weeks for 6 weeks and the 2.9mg/kg dose was administered once every 3 weeks for 6 weeks. Animals weredosed intraperitoneally starting on day 13 post tumor inoculation. Byday 59, the αSA-MCC-DM1 treated group was euthanized due to large tumorvolumes. On day 59, the average tumor volume of mice treated with eachdose regimen of Ab4-MCC-DM1 administered was significantly less thanthat of the αSA-MCC-DM1 control conjugate group (p<0.0001) (FIG. 7).Ab4-MCC-DM1 mediated durable tumor regression with each dosing regimen.No body weight loss was observed in any of the Ab4-MCC-DM1 treatmentgroups.

Pharmokinetics

Ab4 and Ab8 antibody drug conjugates were assessed after intravenousadministration to female CB-17/SCID mice containing CD27L-expressing786-0 xenografts in the context of efficacy studies and a follow-onterminal PK study. The cumulative exposures and clearance values werewithin 1.33 and 1.4-fold between the Ab4-MCC-DM1 and Ab8-MCC-DM1molecules, and the half-life of both ADCs was approximately 12 days inmice.

The stability and PK parameters of the Ab4-MCC-DM1 and Ab8-MCC-DM1antibody drug conjugates were characterized in vivo following a singleintravenous (IV) dose into CB-17/SCID mice bearing 786-0 xenografts. Theexposures of the two antibody drug conjugates were highly comparable,but Ab4-MCC-DM1 demonstrated a more stable drug-to-antibody conjugationratio over the 96 hour time-course of the study as determined byaffinity-MS than did Ab8-MCC-DM1 molecule. Ab4-MCC-DM1 showed nodetectable loss of conjugate integrity at the 30 min or 24 hour timepoint post injection while Ab8-MCC-DM1 exhibited changes at the 30minute time point and significant decreases in all conjugate species by24 hours post injection.

Example 3—Paratope Mapping

Modifications to Ab4 were tested for binding to CD27L by ELISA. CD27Lwas bound to Maxisorp plates at room temperature for >2 hours, shakingwith 1 ug/mL in 100 uL. The plates were then blocked with 250 uL 10%Non-fat dry milk in PBS+0.05% Tween 20 for 2 hours. After a 4×300 uLPBS+0.05% Tween20 (PBST) wash the modified Ab and purified parentalcontrol titrations ranging from 0.045 to 100 ng/mL were applied to theplates and incubated for 1 hour. A horseradish peroxidase conjugatedahuFc detection antibody (Jackson) diluted 1:7000 was applied to theplates after another PBST wash, and incubated for 1 hour. A final washwas performed and TMB substrate was incubated for 10 minutes and stoppedwith phosphoric acid. An OD reading at 450 nm was taken and plotted vs.concentration. These curves were then analyzed with a 3 parameter nonlinear curve fit and the EC₅₀ and Max calculated signal were compared tothe purified control. Binding was determined to be similar to parentalif the EC₅₀ was within 2 fold and the Max signal was greater than 50%.The binding was reduced if the EC₅₀ increased greater than 2 fold and/orthe max signal was less than 50%. The binding was abolished if no curvecould be generated or the max signal was less than 5%.

TABLE 8 Light and heavy chain modifications combinations are listed withtheir expression levels in ug/mL (determined By ForteBio Protein Aquantitation) along with their effect on binding. CombinationsExpression Binding N31H-Y58N + N31S-I34M 30.3 Reduce N31H-Y58N +G33S-I34L 46.7 Abolish N31H-Y58N + D54E-G55S 9.91 Abolish N31H-Y58N +S103G-G104S 14.6 Abolish N31H-Y58N + Parent HC 10.4 Reduce R24K-S26G +N31S-I34M 13.8 Similar R24K-S26G + G33S-I34L 16.9 Abolish R24K-S26G +D54E-G55S 1.87 Reduce R24K-S26G + S103G-G104S 3.76 Reduce R24K-S26G +Parent HC 3.43 Similar L55I-Y58F + N31S-I34M 9.53 Reduce L55I-Y58F +G33S-I34L 11.6 Abolish L55I-Y58F + D54E-G55S 1.26 Reduce L55I-Y58F +S103G-G104S 2.61 Reduce L55I-Y58F + Parent HC 2.72 Similar Q95N-T96S +N31S-I34M 24.6 Similar Q95N-T96S + G33S-I34L 32.7 Abolish Q95N-T96S +D54E-G55S 1.89 Reduce Q95N-T96S + S103G-G104S 4.99 Abolish Q95N-T96S +Parent HC 5.85 Similar Parent LC + N31S-I34M 31.6 Reduce Parent LC +G33S-I34L 51.2 Abolish Parent LC + D54E-G55S 7.03 Reduce Parent LC +S103G-G104S 10.7 Abolish Parent LC + Parent HC 9.25 Similar

13 of the 24 modification combinations made to Ab4 retained some levelof binding to CD27L. The 9 that did not bind to CD27L had at least oneof the following: “G33S-I34L” and “S103G-G104S” heavy chainmodifications or the “N31H-Y58N” light chain modification. Of the 97%similar antibodies (2 amino acid modifications) 75% (6 of 8) retainedsome level of binding, while 56% (7 of 16) of the 94% similar antibodies(4 amino acid modifications) retain some level of binding, 2 of whichhave similar binding to the purified parental control. This shows thatan antibody as low as 94% similar to Ab4 can still bind CD27L.

SEQUENCE LISTING Human CD27L amino acid sequence SEQ ID NO: 1MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP Human CD27 precursor amino acid sequence SEQ ID NO: 2MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP Ab1 Heavy Chain-encoding nucleotide sequenceSEQ ID NO: 3CAGATGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGATGGCTCCATCATCAGTGGTGTTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATCTATTACAGTGGGAGCACCTCCTACAACCCGTCCCTCAAGAGTCGACTTACCATGTCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGGAGTGGATACAGCTATGCCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAb2 Heavy Chain-encoding nucleotide sequence SEQ ID NO: 4CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCATCAGTGGTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTTTTACAGTGGGAGCACCGACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGGAGTGGATACAGCTATGCCCTCTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAb4 Heavy Chain-encoding nucleotide sequence SEQ ID NO: 5CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATACACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGAGGATATAGTGGCTACGATTCGGGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAAb5 Heavy Chain-encoding nucleotide sequence SEQ ID NO: 6CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGTACGATTTTTGGAGTGGTTATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAAb6 Heavy Chain-encoding nucleotide sequence SEQ ID NO: 7CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTTACACACACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACTACGGTGGTAACGACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAAb7 Heavy Chain-encoding nucleotide sequence SEQ ID NO: 8CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAACAGTCACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAb8 Heavy Chain-encoding nucleotide sequence SEQ ID NO: 9CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTGATAAATACTTTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGATAGCAGGAGCTCGCTACGTCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Ab1 Heavy Chain amino acid sequenceSEQ ID NO: 10QMQLQESGPGLVKPSQTLSLTCTVSDGSIISGVYYWSWIRQHPGKGLEWIGYIYYSGSTSYNPSLKSRLTMSVDTSKNQFSLKLSSVTAADTAVYYCARSGYSYALFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab2 Heavy Chain amino acid sequenceSEQ ID NO: 11QVQLQESGPGLVKPSQTLSLTCTVSGDSIISGGYYWSWIRQHPGKGLEWIGYIFYSGSTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSGYSYALFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab4 Heavy Chain amino acid sequenceSEQ ID NO: 12QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSGYDSGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab5 Heavy Chain amino acid sequenceSEQ ID NO: 13QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGYDFWSGYYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab6 Heavy Chain amino acid sequenceSEQ ID NO: 14QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGYTHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYGGNDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab7 Heavy Chain amino acid sequenceSEQ ID NO: 15QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDGSNKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNSHYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab8 Heavy Chain amino acid sequenceSEQ ID NO: 16QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAGARYVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAb1 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 17QMQLQESGPGLVKPSQTLSLTCTVSDGSIISGVYYWSWIRQHPGKGLEWIGYIYYSGSTSYNPSLKSRLTMSVDTSKNQFSLKLSSVTAADTAVYYCARSGYSYALFDYWGQGTLVTVSSAb2 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 18QVQLQESGPGLVKPSQTLSLTCTVSGDSIISGGYYWSWIRQHPGKGLEWIGYIFYSGSTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSGYSYALFDHWGQGTLVTVSSAb3 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 19EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISDSGGTTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHDYSNRYYFDYWGQGTLVTVSSAb4 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 20QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSGYDSGFDYWGQGTLVTV SSAb5 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 21QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGYDFWSGYYYYYYGMDVWGQGT TVTVSSAb6 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 22QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGYTHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYGGNDYYGMDVWGQGTTVTVS SAb7 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 23QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDGSNKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNSHYYYGMDVWGQGTTVTVSSAb8 Heavy Chain Variable Domain amino acid sequence SEQ ID NO: 24QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAGARYVYFDYWGQGTLVTV SSAb1 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 25 SGVYYWSAb2 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 26 SGGYYWSAb3 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 27 SYAMSAb4 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 28 NYGIHAb5 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 29 SYDINAb6 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 30 SYGISAb7 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 31 TYGMHAb8 Heavy Chain CDR1 amino acid sequence SEQ ID NO: 32 SYGMHAb1 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 33 YIYYSGSTSYNPSLKSAb2 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 34 YIFYSGSTDYNPSLKSAb3 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 35 VISDSGGTTDYADSVKGAb4 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 36 VIWYDGSNKYYADSVKGAb5 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 37 WMNPNSGNTGYAQKFQGAb6 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 38 WISAYNGYTHYAQKLQGAb7 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 39 VIWYDGSNKYYGDSVKGAb8 Heavy Chain CDR2 amino acid sequence SEQ ID NO: 40 VIWYDGSDKYFADSVKGAb1 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 41 SGYSYALFDYAb2 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 42 SGYSYALFDHAb3 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 43 HDYSNRYYFDYAb4 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 44 DGGYSGYDSGFDYAb5 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 45 GYDFWSGYYYYYYGMDVAb6 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 46 DYGGNDYYGMDVAb7 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 47 DNSHYYYGMDVAb8 Heavy Chain CDR3 amino acid sequence SEQ ID NO: 48 DGIAGARYVYFDYAb1 Light Chain-encoding nucleotide sequence SEQ ID NO: 49GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTTTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCGTTGACAGATATTTCAATTGGTATCAGCAGAAACCTGGGAAAGCCCCTAAGGTCCTGATCTTTGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCGGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGCTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAb2 Light Chain-encoding nucleotide sequence SEQ ID NO: 50GACATCCAGATGACCCAGTCCCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAGTTGCCGGGCAAGTCAGTTCATTGGCAGATATTTCAATTGGTATCAGCAGCAACCAGGGAAAGCCCCTAAGGTCCTGATCTATGCTGAATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAGATACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAb4 Light Chain-encoding nucleotide sequence SEQ ID NO: 51GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGAATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGTTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGTATACAAACTCTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAb5 Light Chain-encoding nucleotide sequence SEQ ID NO: 52GAAATTGTGTTGACGCAGTCTCCTGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCTGCAGTCTGGTAGCTCTGTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAAAb6 Light Chain-encoding nucleotide sequence SEQ ID NO: 53CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAATTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAGTGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCCTCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAGAb7 Light Chain-encoding nucleotide sequence SEQ ID NO: 54CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTAAATTGGTATCAGCAGTTCCCAGGAACAGCCCCCAAACTCCTCATCTATGTTAACAACAATCGGCCCTCAGGAGTCCCTGACCGATTCTCTGGCTCCACGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACACCAGCCTGAGTGCTTCGGTATTCGGCGGAGGGACCAGACTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCAAb8 Light Chain-encoding nucleotide sequence SEQ ID NO: 55GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAGGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAATATTATAATTACCCATTCACTTTCGGCCCTGGGACCACAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAb1 Light Chain amino acid sequence SEQ ID NO: 56DIQMTQSPSSLSASLGDRVTITCRASQSVDRYFNWYQQKPGKAPKVLIFAASSLQSGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab2 Light Chain amino acid sequenceSEQ ID NO: 57DIQMTQSPSSLSASVGDRVTISCRASQFIGRYFNWYQQQPGKAPKVLIYAESSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFARYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab4 Light Chain amino acid sequenceSEQ ID NO: 58DIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGYNYLDWYLQKPGQSPQFLIYLGSYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCIQTLQTPFTFGPGTKVDIKRTVAAPSVFlFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAb5 Light Chain amino acid sequence SEQ ID NO: 59EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCLQSGSSVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab6 Light Chain amino acid sequenceSEQ ID NO: 60QSVLTQPPSASGTPGQRVTISCSGSSSNIGINYVYWYQQLPGTAPKLLIYRSDQRPSGVPDRFSGSKSGTSASLALSGLRSEDEADYYCAAWDDSLSGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Ab7 Light Chain amino acid sequenceSEQ ID NO: 61QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVNWYQQFPGTAPKLLIYVNNNRPSGVPDRFSGSTSGTSASLAITGLQAEDEADYYCQSYDTSLSASVFGGGTRLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSAb8 Light Chain amino acid sequence SEQ ID NO: 62DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQGGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPFTFGPGTTVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAb1 Light Chain Variable Domain amino acid sequence SEQ ID NO: 63DIQMTQSPSSLSASLGDRVTITCRASQSVDRYFNWYQQKPGKAPKVLIFAASSLQSGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEVKAb2 Light Chain Variable Domain amino acid sequence SEQ ID NO: 64DIQMTQSPSSLSASVGDRVTISCRASQFIGRYFNWYQQQPGKAPKVLIYAESSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFARYYCQQSYSTPWTFGQGTKVEIKAb3 Light Chain Variable Domain amino acid sequence SEQ ID NO: 65EIVLTQSPGTLSLSPGERATLSCRASQSFSSNYLAWYQQKPGQAPRLFIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYGISPCSFGQGTKLEIKAb4 Light Chain Variable Domain amino acid sequence SEQ ID NO: 66DIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGYNYLDWYLQKPGQSPQFLIYLGSYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCIQTLQTPFTFGPGTKVDIKAb5 Light Chain Variable Domain amino acid sequence SEQ ID NO: 67EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCLQSGSSVPLTFGGGTKVEIKAb6 Light Chain Variable Domain amino acid sequence SEQ ID NO: 68QSVLTQPPSASGTPGQRVTISCSGSSSNIGINYVYWYQQLPGTAPKLLIYRSDQRPSGVPDRFSGSKSGTSASLALSGLRSEDEADYYCAAWDDSLSGVVFGGGTKLTVLAb7 Light Chain Variable Domain amino acid sequence SEQ ID NO: 69QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVNWYQQFPGTAPKLLIYVNNNRPSGVPDRFSGSTSGTSASLAITGLQAEDEADYYCQSYDTSLSASVFGGGTRLTVLAb8 Light Chain Variable Domain amino acid sequence SEQ ID NO: 70DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQGGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPFTFGPGTTVDIKAb1 Light Chain CDR1 amino acid sequence SEQ ID NO: 71 RASQSVDRYFNAb2 Light Chain CDR1 amino acid sequence SEQ ID NO: 72 RASQFIGRYFNAb3 Light Chain CDR1 amino acid sequence SEQ ID NO: 73 RASQSFSSNYLAAb4 Light Chain CDR1 amino acid sequence SEQ ID NO: 74 RSSQSLLNSNGYNYLDAb5 Light Chain CDR1 amino acid sequence SEQ ID NO: 75 RASQSVSSSYLAAb6 Light Chain CDR1 amino acid sequence SEQ ID NO: 76 SGSSSNIGINYVYAb7 Light Chain CDR1 amino acid sequence SEQ ID NO: 77 TGSSSNIGAGYDVNAb8 Light Chain CDR1 amino acid sequence SEQ ID NO: 78 RASQGISNYLAAb1 Light Chain CDR2 amino acid sequence SEQ ID NO: 79 AASSLQSAb2 Light Chain CDR2 amino acid sequence SEQ ID NO: 80 AESSLQSAb3 Light Chain CDR2 amino acid sequence SEQ ID NO: 81 GASSRATAb4 Light Chain CDR2 amino acid sequence SEQ ID NO: 82 LGSYRASAb5 Light Chain CDR2 amino acid sequence SEQ ID NO: 83 GASSRATAb6 Light Chain CDR2 amino acid sequence SEQ ID NO: 84 RSDQRPSAb7 Light Chain CDR2 amino acid sequence SEQ ID NO: 85 VNNNRPSAb8 Light Chain CDR2 amino acid sequence SEQ ID NO: 86 AASSLQGAb1 Light Chain CDR3 amino acid sequence SEQ ID NO: 87 QQSYSTPWTAb2 Light Chain CDR3 amino acid sequence SEQ ID NO: 88 QQSYSTPWTAb3 Light Chain CDR3 amino acid sequence SEQ ID NO: 89 QQYGISPCSAb4 Light Chain CDR3 amino acid sequence SEQ ID NO: 90 IQTLQTPFTAb5 Light Chain CDR3 amino acid sequence SEQ ID NO: 91 LQSGSSVPLTAb6 Light Chain CDR3 amino acid sequence SEQ ID NO: 92 AAWDDSLSGVVAb7 Light Chain CDR3 amino acid sequence SEQ ID NO: 93 QSYDTSLSASVAb8 Light Chain CDR3 amino acid sequence SEQ ID NO: 94 QQYYNYPFT

What is claimed is:
 1. An isolated nucleic acid encoding a polypeptide,said polypeptide comprising: a) a light chain variable domaincomprising: i) an LCDR1 comprising the sequence set forth in SEQ ID NO:71; an LCDR2 comprising the sequence set forth in SEQ ID NO: 79; and anLCDR3 comprising the sequence set forth in SEQ ID NO: 87; ii) an LCDR1comprising the sequence set forth in SEQ ID NO: 72; an LCDR2 comprisingthe sequence set forth in SEQ ID NO: 80; and an LCDR3 comprising thesequence set forth in SEQ ID NO: 88; iii) an LCDR1 comprising thesequence set forth in SEQ ID NO: 73; an LCDR2 comprising the sequenceset forth in SEQ ID NO: 81; and an LCDR3 comprising the sequence setforth in SEQ ID NO: 89; iv) an LCDR1 comprising the sequence set forthin SEQ ID NO: 74; an LCDR2 comprising the sequence set forth in SEQ IDNO: 82; and an LCDR3 comprising the sequence set forth in SEQ ID NO: 90;v) an LCDR1 comprising the sequence set forth in SEQ ID NO: 75; an LCDR2comprising the sequence set forth in SEQ ID NO: 83; and an LCDR3comprising the sequence set forth in SEQ ID NO: 91; vi) an LCDR1comprising the sequence set forth in SEQ ID NO: 76; an LCDR2 comprisingthe sequence set forth in SEQ ID NO: 84; and an LCDR3 comprising thesequence set forth in SEQ ID NO: 92; vii) an LCDR1 comprising thesequence set forth in SEQ ID NO: 77; an LCDR2 comprising the sequenceset forth in SEQ ID NO: 85; and an LCDR3 comprising the sequence setforth in SEQ ID NO: 93; viii) an LCDR1 comprising the sequence set forthin SEQ ID NO: 78; an LCDR2 comprising the sequence set forth in SEQ IDNO: 86; and an LCDR3 comprising the sequence set forth in SEQ ID NO: 94;or ix) the amino acid sequence set forth in SEQ ID NO: 66, but having anR24K-S26G mutation, an L551-Y58F mutation, a Q95N-T96S mutation, or anN31H-Y58N mutation; and/or b) a heavy chain variable domain comprising:i) an HCDR1 comprising the sequence set forth in SEQ ID NO: 25; an HCDR2comprising the sequence set forth in SEQ ID NO: 33; and an HCDR3comprising the sequence set forth in SEQ ID NO: 41; ii) an HCDR1comprising the sequence set forth in SEQ ID NO: 26; an HCDR2 comprisingthe sequence set forth in SEQ ID NO: 34; and an HCDR3 comprising thesequence set forth in SEQ ID NO: 42; iii) an HCDR1 comprising thesequence set forth in SEQ ID NO: 27; an HCDR2 comprising the sequenceset forth in SEQ ID NO: 35; and an HCDR3 comprising the sequence setforth in SEQ ID NO: 43; iv) an HCDR1 comprising the sequence set forthin SEQ ID NO: 28; an HCDR2 comprising the sequence set forth in SEQ IDNO: 36; and an HCDR3 comprising the sequence set forth in SEQ ID NO: 44;v) an HCDR1 comprising the sequence set forth in SEQ ID NO: 29; an HCDR2comprising the sequence set forth in SEQ ID NO: 37; and an HCDR3comprising the sequence set forth in SEQ ID NO: 45; vi) an HCDR1comprising the sequence set forth in SEQ ID NO: 30; an HCDR2 comprisingthe sequence set forth in SEQ ID NO: 38; and an HCDR3 comprising thesequence set forth in SEQ ID NO: 46; vii) an HCDR1 comprising thesequence set forth in SEQ ID NO: 31; an HCDR2 comprising the sequenceset forth in SEQ ID NO: 39; and an HCDR3 comprising the sequence setforth in SEQ ID NO: 47; viii) an HCDR1 comprising the sequence set forthin SEQ ID NO: 32; an HCDR2 comprising the sequence set forth in SEQ IDNO: 40; and an HCDR3 comprising the sequence set forth in SEQ ID NO: 48;or ix) the amino acid sequence set forth in SEQ ID NO: 20, but having anN31S-I34M mutation, a D54E-G55S mutation, or an S103G-G104S mutation. 2.The isolated nucleic acid of claim 1, wherein the light chain is encodedby a nucleic acid comprising a nucleotide sequence at least 80%identical to the nucleotide sequence set forth in SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, orSEQ ID NO:
 55. 3. The isolated nucleic acid of claim 2, wherein thelight chain is encoded by a nucleic acid comprising a nucleotidesequence set forth in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO:
 55. 4. The isolatednucleic acid of claim 1, wherein the heavy chain is encoded by a nucleicacid comprising a nucleotide sequence at least 80% identical to thenucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:
 9. 5. Theisolated nucleic acid of claim 4, wherein the heavy chain is encoded bya nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,or SEQ ID NO:
 9. 6. An expression vector comprising the isolated nucleicacid of claim
 1. 7. The expression vector of claim 6, wherein theisolated nucleic acid encodes an antibody light chain.
 8. The expressionvector of claim 6, wherein the isolated nucleic acid encodes an antibodyheavy chain.
 9. The expression vector of claim 7, further comprising anisolated nucleic acid encoding an antibody heavy chain.
 10. Arecombinant host cell comprising the isolated nucleic add of claim 1operably linked to a promoter.
 11. A recombinant host cell comprisingthe expression vector of claim
 9. 12. The recombinant host cell of claim11, wherein the host cell secretes an antibody that binds CD27L.
 13. Therecombinant host cell of claim 12, wherein the cell is of mammalianorigin.
 14. The recombinant host cell of claim 13, wherein the cell is aChinese hamster ovary (CHO) cell.
 15. A method of making a CD27Lantibody drug conjugate, said method comprising the steps of: a)providing a CD27L antigen binding protein encoded by an isolated nucleicacid encoding a polypeptide comprising: i) a light chain variable domaincomprising: i) an LCDR1 comprising the sequence set forth in SEQ ID NO:71; an LCDR2 comprising the sequence set forth in SEQ ID NO: 79; and anLCDR3 comprising the sequence set forth in SEQ ID NO: 87; ii) an LCDR1comprising the sequence set forth in SEQ ID NO: 72; an LCDR2 comprisingthe sequence set forth in SEQ ID NO: 80; and an LCDR3 comprising thesequence set forth in SEQ ID NO: 88; iii) an LCDR1 comprising thesequence set forth in SEQ ID NO: 73; an LCDR2 comprising the sequenceset forth in SEQ ID NO: 81; and an LCDR3 comprising the sequence setforth in SEQ ID NO: 89; iv) an LCDR1 comprising the sequence set forthin SEQ ID NO: 74; an LCDR2 comprising the sequence set forth in SEQ IDNO: 82; and an LCDR3 comprising the sequence set forth in SEQ ID NO: 90;v) an LCDR1 comprising the sequence set forth in SEQ ID NO: 75; an LCDR2comprising the sequence set forth in SEQ ID NO: 83; and an LCDR3comprising the sequence set forth in SEQ ID NO: 91; vi) an LCDR1comprising the sequence set forth in SEQ ID NO: 76; an LCDR2 comprisingthe sequence set forth in SEQ ID NO: 84; and an LCDR3 comprising thesequence set forth in SEQ ID NO: 92; vii) an LCDR1 comprising thesequence set forth in SEQ ID NO: 77; an LCDR2 comprising the sequenceset forth in SEQ ID NO: 85; and an LCDR3 comprising the sequence setforth in SEQ ID NO: 93; viii) an LCDR1 comprising the sequence set forthin SEQ ID NO: 78; an LCDR2 comprising the sequence set forth in SEQ IDNO: 86; and an LCDR3 comprising the sequence set forth in SEQ ID NO: 94;or ix) the amino acid sequence set forth in SEQ ID NO: 66, but having anR24K-S26G mutation, an L551-Y58F mutation, a Q95N-T96S mutation, or anN31H-Y58N mutation; and ii) a heavy chain variable domain comprising: i)an HCDR1 comprising the sequence set forth in SEQ ID NO: 25; an HCDR2comprising the sequence set forth in SEQ ID NO: 33; and an HCDR3comprising the sequence set forth in SEQ ID NO: 41; ii) an HCDR1comprising the sequence set forth in SEQ ID NO: 26; an HCDR2 comprisingthe sequence set forth in SEQ ID NO: 34; and an HCDR3 comprising thesequence set forth in SEQ ID NO: 42; iii) an HCDR1 comprising thesequence set forth in SEQ ID NO: 27; an HCDR2 comprising the sequenceset forth in SEQ ID NO: 35; and an HCDR3 comprising the sequence setforth in SEQ ID NO: 43; iv) an HCDR1 comprising the sequence set forthin SEQ ID NO: 28; an HCDR2 comprising the sequence set forth in SEQ IDNO: 36; and an HCDR3 comprising the sequence set forth in SEQ ID NO: 44;v) an HCDR1 comprising the sequence set forth in SEQ ID NO: 29; an HCDR2comprising the sequence set forth in SEQ ID NO: 37; and an HCDR3comprising the sequence set forth in SEQ ID NO: 45; vi) an HCDR1comprising the sequence set forth in SEQ ID NO: 30; an HCDR2 comprisingthe sequence set forth in SEQ ID NO: 38; and an HCDR3 comprising thesequence set forth in SEQ ID NO: 46; vii) an HCDR1 comprising thesequence set forth in SEQ ID NO: 31; an HCDR2 comprising the sequenceset forth in SEQ ID NO: 39; and an HCDR3 comprising the sequence setforth in SEQ ID NO: 47; viii) an HCDR1 comprising the sequence set forthin SEQ ID NO: 32; an HCDR2 comprising the sequence set forth in SEQ IDNO: 40; and an HCDR3 comprising the sequence set forth in SEQ ID NO: 48;or ix) the amino acid sequence set forth in SEQ ID NO: 20, but having anN31S-I34M mutation, a D54E-G55S mutation, or an S103G-G104S mutation;and b) conjugating a linker covalently bound to a drug to the CD27Lantigen binding protein; or conjugating the CD27 L antigen bindingprotein to a linker, and conjugating a drug to the linker.
 16. Themethod of claim 15, wherein the CD27L antigen binding protein is anantibody.
 17. The method of claim 16, wherein the antibody comprises a)a light chain variable domain amino acid sequence as set forth in SEQ IDNO: 63 and a heavy chain variable domain amino acid sequence as setforth in SEQ ID NO: 17; b) a light chain variable domain amino acidsequence as set forth in SEQ ID NO: 64 and a heavy chain variable domainamino acid sequence as set forth in SEQ ID NO: 18; c) a light chainvariable domain amino acid sequence as set forth in SEQ ID NO: 65 and aheavy chain variable domain amino acid sequence as set forth in SEQ IDNO: 19; d) a light chain variable domain amino acid sequence as setforth in SEQ ID NO: 66 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO: 20; e) a light chain variable domainamino acid sequence as set forth in SEQ ID NO: 67 and a heavy chainvariable domain amino acid sequence as set forth in SEQ ID NO: 21; f) alight chain variable domain amino acid sequence as set forth in SEQ IDNO: 68 and a heavy chain variable domain amino acid sequence as setforth in SEQ ID NO: 22; g) a light chain variable domain amino acidsequence as set forth in SEQ ID NO: 69 and a heavy chain variable domainamino acid sequence as set forth in SEQ ID NO: 23; or h) a light chainvariable domain amino add sequence as set forth in SEQ ID NO: 70 and aheavy chain variable domain amino add sequence as set forth in SEQ IDNO:
 24. 18. The method of claim 17, wherein the antibody comprises a) alight chain amino add sequence as set forth in SEQ ID NO: 56 and a heavychain amino add sequence as set forth in SEQ ID NO: 10; b) a light chainamino acid sequence as set forth in SEQ ID NO: 57 and a heavy chainamino acid sequence as set forth in SEQ ID NO: 11; c) a light chainamino acid sequence as set forth in SEQ ID NO: 58 and a heavy chainamino acid sequence as set forth in SEQ ID NO: 12; d) a light chainamino acid sequence as set forth in SEQ ID NO: 59 and a heavy chainamino acid sequence as set forth in SEQ ID NO: 13; e) a light chainamino acid sequence as set forth in SEQ ID NO: 60 and a heavy chainamino acid sequence as set forth in SEQ ID NO: 14; f) a light chainamino acid sequence as set forth in SEQ ID NO: 61 and a heavy chainamino acid sequence as set forth in SEQ ID NO: 15; or g) a light chainamino acid sequence as set forth in SEQ ID NO: 62 and a heavy chainamino acid sequence as set forth in SEQ ID NO:
 16. 19. The method ofclaim 15, wherein the linker comprises N-succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (MCC).
 20. The method ofclaim 15, wherein the drug comprises maytansinoid DM1.
 21. The isolatednucleic acid of claim 1, wherein said polypeptide comprises: a) a lightchain variable domain comprising at least 95% identity to the amino acidsequence set forth in SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO:70; b) a heavy chain variable domain comprising at least 95% identity tothe amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,or SEQ ID NO: 24; c) a light chain variable domain comprising no morethan five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO: 63, SEQ ID NO: 64, SEQ IDNO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, orSEQ ID NO: 70; or d) a heavy chain variable domain comprising no morethan five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, orSEQ ID NO:
 24. 22. The isolated nucleic acid of claim 1, wherein saidpolypeptide comprises: a) a light chain comprising the amino acidsequence set forth in SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, or SEQ ID NO: 62; and/or b) aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, or SEQ ID NO: 16.